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Effects of Percoll Separation, Cryoprotective Agents, and Temperature on Plasma Membrane Permeability Characteristics of Murine Spermatozoa and Their Relevance to Cryopreservation¹

^a Cryobiology Research Institute, Wells Research Center, Indiana University Medical School, Indianapolis,Indiana 46202 ^b Department of Veterinary Clinical Sciences, School of Veterinary Medicine, Purdue University, West Lafayette,Indiana 47907

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cryopreservation of murine spermatozoa would provide an efficient method for preserving important genotypes. However, to date such methods have resulted in low survivals with significant variability. To address this issue, a series of five experiments was performed to determine the cryobiological characteristics of murine spermatozoa. Experiments 1 and 2 investigated the effect of Percoll separation on the hydraulic conductivity (L_p) of murine spermatozoa. Both Percoll separation and cryoprotective agents (CPAs) decreased the L_p. However, these effects were not additive. Experiment 3 was performed to determine the effect of temperature on L_p in the presence of cryoprotectants (L_p^CPA), cryoprotectant permeability (P_CPA), and the reflection coefficient () in spermatozoa from both ICR and B6C3F1 mice. Permeability parameters decreased as temperature decreased, and permeability characteristics differed between strains. In experiments 4 and 5, theoretical simulations for CPA addition and removal were developed and empirically tested. Strain-specific methods for CPA addition and removal based upon the fundamental cryobiological characteristics of murine spermatozoa resulted in higher survivals than current methods or procedures, which were used as controls.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The first reports of successful development of transgenic mice were made in the early 1980s [1, 2], and subsequent reports of the development of gene knockout mice were made later in that decade [3, 4]. Since that time, the creation of new strains of mice has been increasing at an exponential rate [5]. The costs associated with housing these new strains of mice are rapidly mounting and are placing a tremendous burden on institutional budgets and infrastructure. It is becoming a practical and economic necessity to find an effective method for preserving and archiving these unique and valuable genetic resources.

Successful cryopreservation of murine embryos in 1971 provided an alternative to maintaining mouse strains in breeding colonies [6]. Subsequently, many laboratories have succeeded in freezing murine embryos, and cryopreservation of murine embryos is routinely performed; however, this method is relatively costly and time consuming. Successful cryopreservation of murine spermatozoa would greatly decrease initial costs as well as long-term labor for murine line preservation.

One particular difficulty in establishing fundamental information regarding murine spermatozoa is that these cells are mechanically sensitive [7, 8] (e.g., vulnerable to various collection, handling, and isolation procedures), a fact that makes it very difficult to obtain a pure sample for experimentation. In order to solve this problem, a density gradient (e.g., Percoll) is often used to purify murine spermatozoa and maximize motility. However, Tanphaichitr et al. [9] reported that using Percoll to purify murine spermatozoa reduces mouse plasma membrane phospholipid levels by 50%. The reduction of phospholipid membrane levels could have significant consequences on the fundamental cryobiological properties of these cells and thereby alter the cells' response to the cryopreservation process.

Since 1990 there have been several reports of successful cryopreservation of murine spermatozoa [10–18]. However, some laboratories have had difficulty repeating published freezing protocols [15, 19]. There have also been reports of strain-to-strain variability with respect to given protocols [18], in that a given procedure may work well for one strain and not at all for another. Freezing protocols developed for certain cell types should be reliable and repeatable. Therefore, it is our thesis that an improved understanding of fundamental cryobiological properties of murine spermatozoa is required to establish optimal freezing protocols that could be employed for all strains of mice.

Knowledge of fundamental cryobiological properties of cells includes an understanding of the osmotically inactive fraction of the cell volume (V_b), permeability of the cell to water (L_p) and cryoprotective agents (P_CPA) as well as the potential interaction of water and cryoprotective agent (CPA) fluxes (i.e., reflection coefficient; ), and the temperature dependence of L_p and P_CPA (i.e., activation energies; E_a). Through the use of the Boyle-van't Hoff relationship (a plot of cell volume vs. 1/osmolality), Willoughby et al. [20] determined that 61% (V_b = 0.61) of the cell volume of murine sperm is composed of solids and nonosmotically active water while 39% of the cell volume is composed of osmotically active water. Furthermore, Willoughby et al. [20] determined that to maintain > 85% functional survival (motility) of murine sperm, cell volume excursions must be kept between 91% and 110% of normal isosmotic volume during the process of cryopreservation (e.g., during CPA addition and removal). Noiles et al. [21] determined that the E_a of L_p in the absence of CPAs for CD-1 mice was 11–14.2 Kcal/mol. However, Gilmore et al. [22, 23] have reported a significant reduction of L_p and an increase in E_a of L_p in the presence of CPAs (L_p^CPA) for other mammalian spermatozoa (e.g., human and boar). Therefore, L_p^CPA and P_CPA for murine spermatozoa need to be determined. Based upon these fundamental cryobiological parameters, improved methods for cryopreservation can be established.

The objectives of the present work were to 1) compare the L_p values for spermatozoa that have been either prepared with Percoll or washed, 2) compare the effects of adding CPA to Percoll-prepared cells and washed cells, 3) create Arrhenius plots for the L_p^CPA and P_CPA values to determine activation energies for murine spermatozoa of both ICR and B6C3F1 mice, 4) develop methods for CPA addition based upon fundamental data, and 5) develop methods for CPA removal based upon fundamental data.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Media

All chemicals were obtained from Sigma Chemical Company (St. Louis, MO) unless otherwise stated. The spermatozoa collection medium used was similar to that described previously by Willoughby et al. [20]. The medium consisted of Dulbecco's PBS (Gibco BRL, Life Technologies, Grand Island, NY) supplemented with DL-lactic acid (2 µl/ml) and 0.4% BSA. This modified supplemented-PBS (S-PBS) solution was filtered with a 0.45-µm acrodisc (Gelman Sciences Inc., Ann Arbor, MI). Milli-Q water (Millipore, Bedford, MA) was added to isosmotic PBS to make 150 mOsm PBS. The CPA solutions were prepared by mixing ethylene glycol (EG), glycerol, 1,2-propanediol (PG), or dimethyl sulfoxide (DMSO) with isosmotic PBS to yield a final concentration of 2.0 M. In experiments 1–3, various 2.0 M CPA solutions were added slowly to sperm-containing solutions to yield a final concentration of 0.5 M (DMSO and glycerol) or 1.5 M (glycerol, EG, and PG).

Spermatozoa Collection and Processing

Male ICR and B6C3F1 mice (Harlan Sprague Dawley, Indianapolis, IN) between 8 and 20 wk of age were used for the experiments. Two to four mice from each strain were killed using isoflurane overdose, followed by cervical dislocation. The cauda epididymides were excised, placed in a 35 x 10-mm Falcon (Falcon Plastics, Los Angeles, CA) dish containing 500 µl modified S-PBS solution at 37°C, then stripped and minced to release sperm cells. Samples were allowed to incubate for 5 min at 37°C and were then pooled in a 15-ml conical tube yielding 1–2 ml of a spermatozoa suspension, including modified S-PBS solution. Cell suspensions that were to be used for non-Percoll-prepared experiments were washed three times before use (centrifuged at 350 x g for 7 min and resuspended in S-PBS solution). Cell suspensions to be used for Percoll-prepared experiments were gently mixed for 5 min, layered on a discontinuous 90%/45% Percoll gradient in a ratio of 1:1:1 ml (cell sample : 45% Percoll : 90% Percoll), and centrifuged at 500 x g for 13 min to remove nonmotile sperm, blood cells, and epithelial cells. The cells separated into three distinct layers. The top two layers were discarded (containing debris, non-sperm cells, and immotile cells); the bottom pellet (containing motile spermatozoa) was washed with the modified S-PBS solution and then centrifuged at 350 x g for 7 min. The supernatant was removed, leaving approximately 0.5 ml of cells per 15-ml conical tube.

Electronic Particle Counter

An electronic particle counter (EPC) approach was used for determining membrane permeability characteristics [22]. Briefly, a ZM model Coulter Counter (Coulter Counter Electronics, Inc., Hialeah, FL) with a 50.0-µm high-resolution aperture tube was used for measurements. An EPC was interfaced to a microcomputer using a CSA-2S interface (Great Canadian Computer Company, Edmonton, AB, Canada). Data at 22°C were obtained by allowing spermatozoa and filtered PBS solution (290 or 150 mOsm, depending on experiment) to equilibrate to room temperature. Cells, with an average concentration of 10 million cells/ml, were then introduced into a 10-ml vial containing PBS solution. The volume of cell suspension introduced into the cup was dependent upon the cellular concentration. This method was used for all data obtained at 22°C. Temperatures below 22°C were obtained through the use of a water bath and water-jacketed beaker. Filtered media, spermatozoa, vials, and aperture tube were all cooled to the desired temperature before the start of the experiment. After reaching the desired temperature, cells were introduced into the vials, as was done at 22°C.

Membrane Permeability Coefficients

A model of water and solute transport based upon irreversible thermodynamics was used to determine murine sperm permeability parameters. Specifically, one pair of coupled nonlinear equations introduced by Kedem and Katchalsky [24] was used as the theoretical model 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:

and

where V is cell volume and mⁱ_s is the molal concentration of CPA inside the cell, and

The superscripts "i" and "e" refer to the intra- and extracellular cell compartment, respectively. The terms L_p^CPA, P_CPA, and are parameters for the hydraulic conductivity of water (in the presence of CPA), the permeability coefficient of the CPA, and the reflection coefficient, respectively. The temperature and universal gas constant are given by T and R, respectively. For the impermeable solute (NaCl), the intracellular osmolality of NaCl is given by:

where V_b is the osmotically inactive cell volume and is the CPA volume ( is the partial molar volume of the CPA). The superscript "(o)" represents the initial values at t = 0. The values for L_p^CPA, P_CPA, and were determined. A fixed value for V_b [20] was used in the analysis.

Activation Energies for Parameters L_p and P_CPA

The Arrhenius relationship was used to determine the activation energies of the parameters L_p^CPA and P_CPA [25]. The permeability value (L_p^CPA or P_CPA) at any temperature T can be plotted as ln[P_a(T)] vs. 1/T, (Arrhenius plot),

where E_a is the activation energy for the process, expressed in Kcal/mol. The slope of the plot is defined as:

from which E_a can be determined. Definitions of all variables used above are presented in Chart 1.

Theory of Optimal CPA Addition and Dilution

Methods for optimal addition and dilution are defined here as the processes that minimize the number of addition and dilution steps as well as osmotic cell volume excursion. The theoretical considerations and flow diagram for this procedure are described by Gilmore et al. [26]. Computer simulations were designed to predict optimal CPA addition and removal procedures from murine spermatozoa. Under the given experimental conditions (initial intracellular concentration, upper and lower osmotic tolerance limits of the cell, and temperature), the program optimizes the addition and dilution steps automatically and provides the appropriate diluent concentration. Figure 1 illustrates the relative cell volume of mouse spermatozoa (ICR in panel A and B6C3F1 in panel B) as a function of time when 1 M glycerol, EG, or PG is added. Figure 2 illustrates the relative cell volume of mouse spermatozoa (ICR mice in panel A and B6C3F1 mice in panel B) as a function of time when 1 M glycerol, EG, or PG is removed.

View larger version (18K): [in this window] [in a new window]   FIG. 1. Theoretical simulation for the 1-step addition of 1 M glycerol (dashed/dotted line), EG (solid line), and propylene glycol (PG) (dotted line) to spermatozoa from ICR mice (A) and B6C3F1 mice (B) at 22°C

View larger version (17K): [in this window] [in a new window]   FIG. 2. Theoretical simulation for the 1-step removal of 1 M glycerol (dashed/dotted line), EG (solid line), and propylene glycol (PG) (dotted line) from ICR (A) or B6C3F1 (B) murine spermatozoa at 22°C

Statistical Analysis

Standard ANOVA procedures were implemented using General Linear Models procedure in the Statistical Analysis Software (SAS) program. Log transformation procedures were performed when data were not normally distributed, and differences were considered statistically significant if P < 0.05.

In experiments 4 and 5, the percentage of motile spermatozoa was measured after each treatment and was normalized to an untreated control (treatment motility/control motility x 100). The data are presented as normalized values. The mean and SE were computed for each treatment, and the data were analyzed using standard ANOVA with the SAS program.

Experimental Design

Experiment 1: Effect of Percoll separation on hydraulic conductivity (L_p) of murine spermatozoa Spermatozoa were collected from B6C3F1 mice. The cells were either processed on a Percoll gradient or washed in S-PBS solution. Hydraulic conductivity values were determined through the use of an EPC by exposing cells in an isosmotic PBS solution to a PBS solution of 150 mOsm at 22°C. Hydraulic conductivities of the Percoll-prepared and washed spermatozoa were then compared to determine whether the use of Percoll significantly reduced L_p. A minimum of four pooled samples (n = 4) and four replicates within a pooled sample were performed at each temperature.

Experiment 2: Effect of Percoll separation on hydraulic conductivity (L_p^CPA) of murine spermatozoa in the presence of CPAs Spermatozoa were collected from B6C3F1 mice. The cells were either processed on a Percoll gradient or washed in S-PBS solution. A CPA (DMSO or glycerol) was then added to the cells of each treatment to yield a final concentration of 0.5 M. Hydraulic conductivity values were determined through the use of an EPC by exposing cells in 0.5 M CPA to isosmotic solution at 22°C. Hydraulic conductivities of Percoll-prepared plus CPA and washed samples plus CPA were compared to determine whether Percoll-prepared cells differed in their L_p^CPA values from washed cells. A minimum of four pooled samples (n = 4) and four replicates within a pooled sample were performed at each temperature. Due to the low permeability of DMSO and its associated L_p^CPA, and therefore its potential for inducing damaging volume excursions on murine spermatozoa, it was excluded from the subsequent experiments.

Experiment 3: Effect of temperature on hydraulic conductivity (L_p^CPA), the permeability coefficient for the cryoprotectant solute (P_CPA), and the reflection coefficient () Spermatozoa from B6C3F1 and ICR mice were collected and processed on a Percoll gradient. CPA (100 µl) was added drop-wise over 60 sec to 100 µl of cell suspension, and samples were brought to the desired temperature. Cells were allowed to equilibrate for approximately 3 min before all 200 µl was returned to isosmotic media. An EPC measured changes in cell volume over time at different temperature points (22, 15, 10, 5, 1.5, and -2°C). The values for L_p^CPA and P_CPA were plotted on an Arrhenius plot, and activation energies were obtained for these membrane parameters. A minimum of four pooled samples (n = 4) and four replicates within a pooled sample was analyzed at each temperature.

Experiment 4: Addition of CPA to murine spermatozoa Glycerol and EG were investigated here to empirically test methods for CPA addition (and CPA removal in experiment 5). 1,2-Propanediol, having permeability characteristics similar to those of glycerol, was excluded from this study. Two approaches were used for the addition of 2 M glycerol and 2 M EG to the spermatozoa suspension (both B6C3F1 and ICR), yielding final concentrations of 1 M or 0.5 M CPA. In the first approach, 100 µl of a given CPA was added using a fixed molar method as described in Chart 2. The percentage motility was then assessed. The second approach was used as a control and was performed by abruptly adding CPA (using volumes specified in Chart 2) to 100 µl of cell suspension and then assessing motility. Addition of a CPA was performed at 22°C for both the experimental treatment and the control. Three replicates were performed for each strain.

Experiment 5: Removal of CPA from murine spermatozoa Two approaches were used for the removal of 1 M or 0.5 M glycerol or EG from mouse spermatozoa. In the samples to which CPA was added using a fixed molar approach, the same approach was implemented to remove the CPA, as described in Chart 2. In the second approach, the control, CPA was added abruptly, and dilution was done abruptly using volumes of isosmotic PBS described in Chart 2. Motility was measured after CPA dilution. Cryoprotectant removal was performed at 22°C. Three replicates were performed for each strain.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Experiment 1: Effect of Percoll Separation on Hydraulic Conductivity (L_p) of Murine Spermatozoa

The L_p value for spermatozoa that were washed was 0.84 ± 0.08 µm·min^-1·atm^-1 (mean ± SEM; n = 5). The L_p was reduced (P = 0.0007) by 35% when samples were separated by the use of a Percoll gradient (0.56 ± 0.03 µm·min^-1·atm^-1).

Experiment 2: Effect of Percoll Separation on Hydraulic Conductivity (L_p^CPA) of Murine Spermatozoa in the Presence of CPAs

The L_p^CPA values for spermatozoa processed by the various methods were as follows. Percoll processed plus 0.5 M DMSO was 0.47 ± 0.04 µm·min^-1·atm^-1 (mean ± SEM; n = 4); washed plus 0.5 M DMSO was 0.34 ± 0.05 µm·min^-1·atm^-1 (n = 4); Percoll processed plus 0.5 M glycerol was 0.41 ± 0.03 µm·min^-1·atm^-1 (n = 4); and washed plus 0.5 M glycerol was 0.44 ± 0.02 µm·min^-1·atm^-1 (n = 4). Data analysis indicated that there was neither a main effect of Percoll (P = 0.27), CPA (P = 0.47) nor a Percoll-by-CPA interaction (P = 0.11) on hydraulic conductivity. When CPA was added to either Percoll-separated or washed cells, there was not a significant effect (P = 0.27) of Percoll in reducing L_p. These data indicate that the addition of CPA to Percoll-prepared cells does not create an additive effect in reducing L_p. There was no significant difference (P = 0.47) between using 0.5 M DMSO and using 0.5 M glycerol. Furthermore, there was no significant (P = 0.11) effect of CPA on Percoll-separated cells.

Experiment 3: Effect of Temperature on Hydraulic Conductivity (L_p^CPA), the Permeability Coefficient for the Cryoprotectant Solute (P_CPA), and the Reflection Coefficient ()

The results for L_p^CPA, P_CPA, and from experiment 3 are shown in Table 1 (ICR) and Table 2 (B6C3F1).

Hydraulic conductivity (L_p^CPA) For spermatozoa from ICR mice at 22°C, the CPAs EG and glycerol had equally high L_p^CPA values and PG had a lower value. In spermatozoa from B6C3F1 mice, L_p^EG L_p^glycerol > L_p^PG. There was no main effect (P = 0.19) of strain, nor was there a strain-by-CPA interaction (P = 0.49) on L_p^CPA. The type of CPA and temperature did have significant effects (P = 0.03 and 0.0001, respectively) on the L_p^CPA of spermatozoa from both ICR and B6C3F1 mice.

Effect of temperature on hydraulic conductivity (E_a for L_p^CPA) Using temperature points ranging from 22°C to -2°C, activation energies (E_a) for L_p^CPA (in the presence of EG, glycerol, and PG) for spermatozoa from ICR mice were 6.60 (r² = 0.61), 8.72 (r² = 0.80), and 6.95 (r² = 0.72) Kcal/mol, respectively (Table 3). The Arrhenius plots consistently showed deviations from a linear pattern at 15°C and 1.5°C, as shown in Figures 3 and 4, respectively. Therefore, activation energies were determined with these two temperature points excluded (Table 3). The E_as for L_p^EG (Fig. 3), L_p^glycerol (Fig. 4), and L_p^PG were 7.09 (r² = 0.99), 8.48 (r² = 0.87), and 6.44 (r² = 0.66) Kcal/mol, respectively (Table 3). When all temperature points were used to calculate E_a (Table 3) for L_p^CPA (in the presence of EG, glycerol, and PG) of spermatozoa from B6C3F1 mice, the results were 7.37 (r² = 0.77), 8.01 (r² = 0.17), and 1.23 (r² = 0.02) Kcal/mol, respectively. When the values at 15°C and 1.5°C were excluded, the E_as for L_p^EG (Fig. 3), L_p^glycerol (Fig. 4), and L_p^PG were 14.29 (r² = 0.77), 14.92 (r² = 0.74), and 5.89 (r² = 0.55) Kcal/mol (Table 3).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Arrhenius plot of LpEG for A) ICR spermatozoa measured at 22, 9, 4, and -2°C and B) B6C3F1 spermatozoa measured at 22, 10, 5, and -2°C (filled circles, left to right). The temperature points 1.5 and 15°C are shown (open circles), but were not used to determine Ea

View larger version (16K): [in this window] [in a new window]   FIG. 4. Arrhenius plot of Lpglycerol for A) ICR spermatozoa measured at 22, 9, 4, and -2°C and B) B6C3F1 spermatozoa measured at 22, 10, 5, and -2°C. The temperature points 1.5 and 15°C are shown (open circles), but were not used to determine Ea

Permeability coefficient for the cryoprotectant (P_CPA) For spermatozoa from ICR mice at 22°C, P_EG > P_glycerol > P_PG (P < 0.05) (Table 1). For spermatozoa from the B6C3F1 strain at 22°C, P_glycerol > P_EG > P_PG (P < 0.05) (Table 2). Strain, CPA, and temperature each had a main effect (P = 0.0001 for all three) on the P_CPA. There were also strain-by-CPA (P = 0.002), strain-by-temperature (P = 0.001), and CPA-by-temperature (P = 0.008) interactions.

Effect of temperature on permeability coefficient (E_a for P_CPA) When temperature points ranging from 22°C to -2°C were used to determine the E_a of P_CPA (Table 3) of spermatozoa from ICR mice for EG, glycerol, and PG, the results were 11.88 (r² = 0.70), 11.09 (r² = 0.76), and 6.39 (r² = 0.35) Kcal/mol, respectively. As with the L_p^CPA values, the Arrhenius plots consistently showed deviations from a linear pattern at 15°C and 1.5°C (Figs. 5 and 6). Therefore, E_a of P_CPA values were determined with these two temperature points excluded (Table 3). When temperatures 15°C and 1.5°C were excluded, the E_as for P_EG (Fig. 5), P_glycerol (Fig. 6), and P_PG were 10.73 (r² = 0.79), 9.16 (r² = 0.75), and 5.84 (r² = 0.61) Kcal/mol, respectively. When all temperature points were used to calculate E_a of P_CPA (Table 3) of spermatozoa from B6C3F1 mice for P_EG, P_glycerol, and P_PG, the results were 5.36 (r² = 0.40), 8.44 (r² = 0.35), and 2.80 (r² = 0.26) Kcal/mol, respectively. When values from 15 and 1.5°C were excluded, the E_as for P_EG (Fig. 5), P_glycerol (Fig. 6), and P_PG were 8.17 (r² = 0.84), 13.17 (r² = 0.79), and 4.55 (r² = 0.61) Kcal/mol, respectively.

View larger version (17K): [in this window] [in a new window]   FIG. 5. Arrhenius plot of PEG for A) ICR spermatozoa measured at 22, 9, 4, and -2°C and B) B6C3F1 spermatozoa measured at 22, 10, 5, and -2°C. The temperature points 1.5 and 15°C are shown (open circles), but were not used to determine Ea

View larger version (17K): [in this window] [in a new window]   FIG. 6. Arrhenius plot of Pglycerol for A) ICR spermatozoa measured at 22, 9, 4, and -2°C and B) B6C3F1 spermatozoa measured at 22, 10, 5, and -2°C. The temperature points 1.5 and 15°C are shown (open circles), but were not used to determine Ea

Reflection coefficient () Reflection coefficient values can be seen in Table 2. There was a main effect of temperature (P = 0.0001) and CPA (P = 0.0001) on ; however, there was not a main effect of strain (P = 0.31) on .

Experiment 4: Addition of CPA to Murine Spermatozoa

The percentage of motile murine spermatozoa was calculated after aliquots of cell samples were exposed to either glycerol or EG at 22°C using a fixed molar approach or an abrupt approach (ICR mice in Fig. 7; B6C3F1 mice in Fig. 8), yielding final concentrations of 1 M or 0.5 M CPA. There was a main effect of CPA type (P = 0.0001) and CPA concentration (P = 0.0001) on motility. In addition, fixed molar methods for CPA addition resulted in significantly higher (P = 0.0001) motilities than abrupt (control) methods.

View larger version (34K): [in this window] [in a new window]   FIG. 7. Normalized percent motility recovery (mean ± SEM) of spermatozoa from ICR mice after a fixed molar approach (optimized approach) to glycerol (GLY) or ethylene glycol (EG) addition (all treatments), and before and after slow CPA removal (- or +, respectively) (A). Control treatments are shown in B and show motility recovery after abrupt glycerol or EG addition (all treatments) and before and after abrupt CPA removal (- or +). Comparisons between optimized and control methods are between panels within treatment group

View larger version (33K): [in this window] [in a new window]   FIG. 8. Normalized percent motility recovery (mean ± SEM) of spermatozoa from B6C3F1 mice after a fixed molar approach (optimized approach) to glycerol (GLY) or ethylene glycol (EG) addition (all treatments), and before and after slow CPA removal (- or +, respectively) (A). Control treatments are shown in B and show motility recovery after abrupt glycerol or EG addition (all treatments) and before and after abrupt CPA removal (- or +). Comparisons between optimized and control methods are between panels within treatment group

Experiment 5: Removal of CPA

Motility recovery was measured after 1 M or 0.5 M glycerol or EG was slowly or abruptly removed from the spermatozoa (ICR in Fig. 7; B6C3F1 in Fig. 8). Fixed molar methods for CPA removal resulted in significantly higher (P = 0.0001) motilities than abrupt (control) methods.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Effect of Percoll Separation on Hydraulic Conductivity (L_p) of Murine Spermatozoa

Percoll density gradient separation is a common and fairly successful method used to isolate viable murine spermatozoa. However, Tanphaichitr et al. [9] reported that use of the Percoll technique reduces the plasma membrane phospholipid levels in the mouse by one half. Data from the current work (experiment 1) indicate that Percoll separation significantly (P = 0.0007) decreases the L_p of murine spermatozoa when compared to the L_p of spermatozoa that have not been Percoll separated. These findings suggest the possible connection between plasma membrane phospholipid levels and mass transport parameters. These findings also imply that investigators should consider the effects of isolation methodologies on subsequent applications such as cryopreservation.

Effect of Percoll Separation on Hydraulic Conductivity (L_p) of Murine Spermatozoa in the Presence of CPAs

This experiment was performed to determine the effect of CPA addition on L_p of both Percoll- and non-Percoll-separated cells. These data indicate that the two CPAs reduce L_p to approximately the same extent, but that these effects are not additive. Therefore, using Percoll separation in combination with permeating CPAs should not introduce a confounding problem in terms of subsequent cryopreservation processes (e.g., cooling rate). However, with use of Percoll separation in combination with freezing media that do not contain permeating CPAs (e.g., skim milk and sugar), such confounding effects might occur.

Effect of Temperature on Hydraulic Conductivity (L_p), the Permeability Coefficient for the Cryoprotectant Solute, and the Reflection Coefficient ()

Frequently, membrane permeability is measured as a function of temperature; this temperature dependence of transport parameters is often expressed as the activation energy. Knowledge of the activation energy of a transport parameter (either L_p or P_CPA) can be used to optimize cooling and warming rates and can also be used to determine the mechanism of transport (e.g., bilayer transport or facilitated diffusion).

There was a significant effect (P < 0.05) of temperature and CPA type on the hydraulic conductivity of murine spermatozoa. In addition, the E_a of L_p^CPA was different for spermatozoa from ICR and B6C3F1 mice. These data support prior observations by Songsasen and Leibo [18] that a successful freezing protocol developed for one strain of mouse may be inefficient in another strain.

Values reported here for L_p^CPA and P_CPA are similar to those reported for other cell types in which activation energies have been determined using suprazero temperatures [22, 23]. However, these data are very different from findings in those studies in which activation energies have been derived for mouse spermatozoa using subzero temperatures [27]. These data from the present study also indicate that the Arrhenius relationships of L_p^CPA and P_CPA deviate from a linear pattern with a change in temperature. Some temperature points on the plot show a marked deviation from the expected linear response; however, there does appear to be a fairly consistent pattern. The points that appear to deviate the most are data from 15°C and 1.5°C. If these two temperature points are omitted, the goodness-of-fit is better (increased r² values). Omitting these two temperature points has little effect on E_a for spermatozoa from ICR mice. For spermatozoa from B6C3F1 mice, however, omitting these two temperature points significantly increased the r² values and nearly doubled the estimated E_as.

One possible explanation for the lack of goodness-of-fit in the linear regressions when all temperatures were used to create Arrhenius plots is that those temperatures may be associated with lipid phase transitions in the plasma membrane. Phase transitions occurring over a short, suprazero temperature range have been described in spermatozoa of a number of species including the boar, shrimp, and goat [28], while a phase transition occurring over a broader temperature range has been reported in human sperm [29]. Noiles et al. [30] reported a possible membrane phase transition between 4°C and 0°C in murine spermatozoa. In the Arrhenius plots generated from the current data, two of the nonlinear points indicated a decrease in water flow across the membrane (i.e., a decrease in L_p). While previous reports [31] have suggested that at the point of a phase transition, L_p could increase, it seems likely that the plasma membrane is so complex that it is difficult to predict what effect lipid phase transitions would have on membrane permeability characteristics. It is also likely that multiple phase transitions are taking place within these complex plasma membranes.

Addition and Removal of CPAs

Cryopreservation consists of multiple steps, including CPA addition and subsequent removal, CPA permeability to the membrane, cooling and warming rates (including water exosmosis from the cell), and type and concentration of CPA. Previous work has been performed to investigate various methods for CPA addition and removal from mouse spermatozoa, and data support the concept that mouse spermatozoa are extremely sensitive to osmotic changes [32].

Using permeability coefficients characterized in the present study, theoretical models were employed to predict the response of murine spermatozoa to the addition and removal of glycerol, EG, and PG. The theoretical data indicate that in most cases, multiple steps are required for the addition and dilution of cryoprotective solutes from spermatozoa of both ICR and B6C3F1 mice to prevent osmotic damage (Chart 2). These current data show that the number of steps for CPA addition and removal are CPA dependent. For example, 1 M EG can be added to ICR strain mouse spermatozoa using a 1-step fixed molar approach, whereas addition of glycerol requires a 2-step fixed molar approach to prevent osmotic damage. Of equal importance, the data also show that the number of addition and dilution steps is dependent upon the strain. Theoretical predictions indicate that spermatozoa from ICR mice should tolerate a 1-step fixed molar addition of 1 M EG, whereas spermatozoa from B6C3F1 mice require a minimum of 3 fixed molar steps for EG addition. Again, these data support the hypothesis that spermatozoa from different murine genotypes will require unique cryopreservation protocols.

These theoretical predictions were empirically tested, and overall, those results indicated that greater survival was obtained in mouse spermatozoa when a step-wise method for CPA addition and dilution is used (Figs. 7 and 8). However, even with the use of fixed molar methods for CPA addition and removal, significant cell loss occurred with certain treatments (i.e., slow dilution of 1 M glycerol resulted in only 18.3% normalized motility in ICR spermatozoa). These data indicate that osmotic damage may not be the sole cause for injury during CPA addition and removal in murine spermatozoa. Prior studies in other cell types have suggested damage due to water flux stress [31]. In such cases, the pressure that develops during certain anisosmotic conditions may be sufficient to cause a rupture of the plasma membrane due to excessive water flux [31]. Water flux stress may be another contributor to mouse spermatozoa damage during CPA addition and removal.

The data from the current study can be used to gain insight into the results and interpretation of those previously published by Storey et al. [32]. Storey et al. [32] investigated procedures for CPA (6% glycerol) addition and removal (serial addition/dilution and dialysis addition/dilution) in the presence of a sugar (trehalose) from murine spermatozoa. Data from their study support the data from the current study suggesting that a step-wise method for addition/dilution results in relatively high cell viability. However, Storey et al. [32] ultimately concluded that a method incorporating a dialysis membrane for infinitely slow CPA addition, and a method that does not remove CPA prior to insemination (CPA is removed in 1 step), is superior to step-wise methods for addition/dilution (resulting in the highest percentage of in vitro fertilization).

Using the data obtained from the present study, a model of that dialysis membrane system (using permeability coefficients known for urea and assuming those coefficients are similar for glycerol) and the 8-step addition/dilution method used by Storey et al. [32] was developed (Fig. 9). The model shows that using either approach, the same final spermatozoa volume excursions occur, although the time course is very different for the different approaches. Storey et al. [32] found that higher in vitro fertilization was obtained after a 1-step dilution process; however, the authors failed to emphasize that such dilution was made in the presence of a sugar that functions as an "osmotic buffer" to maintain cell volumes within their osmotic tolerance limits (cells are somewhat dehydrated before the dilution process, and therefore volumes do not exceed the upper osmotic tolerance limit when glycerol is removed). Data from the present study suggest that in the absence of hyperosmotic conditions prior to dilution (e.g., sugars), multiple dilution steps are required to maintain the cells within their osmotic tolerance limits and maximize cell survival.

View larger version (17K): [in this window] [in a new window]   FIG. 9. Addition (A) and dilution (B) of 6% glycerol to murine spermatozoa using a 1-step method, step-wise method, and a dialysis method based upon previous reports [32]. Horizontal lines represent lower osmotic tolerance limits of murine spermatozoa in which 85% motility is maintained. The upper osmotic tolerance limits are not shown (off range)

Murine sperm cryopreservation could be significantly improved by broadening the osmotic tolerance limits of the cells and minimizing the damage incurred by the cells during volume excursions. Currently, we are attempting to identify the effects of extenders on the plasma membrane characteristics of mouse sperm. Preliminary data suggest that murine spermatozoa are more osmotically tolerant in the presence of skim milk or egg yolk.

In conclusion, the L_p values for spermatozoa are lower for Percoll-prepared versus non-Percoll-prepared samples (although there is no additive effect of CPA), and therefore methodologies for sperm separations should be considered when one is developing cryobiological protocols. Determining fundamental cryobiological characteristics of murine spermatozoa is effective in developing protocols for cryopreservation. Because these characteristics are strain dependent, optimal cryopreservation protocols will likely be different for different genotypes. Specifically, to optimize murine spermatozoa cryopreservation protocols across strains, fundamental cryobiological parameters should be determined for each strain of interest.

	FOOTNOTES

 
¹ This work was supported by the Cryobiology Research Institute and grants from the NIH (K04-HD00980, R01-HD30274, and R24-RR13194).

² Correspondence: John K. Critser, Cryobiology Research Institute, Cancer Research Building, Wells Research Center, Room 454, 1044 West Walnut Street, Indianapolis, IN 46202. FAX: 317 274 8679; jcritser{at}iupui.edu

Accepted: May 26, 1999.

Received: March 16, 1999.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Brinster RL, Chen HY, Trumbauer M, Senear AW, Warren R, Palmiter RD. Somatic expression of herpes kinase following injection of fusion gene into eggs. Cell 1981; 27:223–231.[CrossRef][Medline]
2. Brinster RL, Palmiter RD. Introduction of genes into the germ line of animals. Harvey Lect 1984; 80:1–38.[Medline]
3. Viney JL. Transgenic and knockout models for studying diseases of the immune system. Curr Opin Genet Dev 1994; 4:461–465.[CrossRef][Medline]
4. Waldman AS. Targeted homologous recombination in mammalian cells. Crit Rev Oncol-Hematol 1992; 12:49–64.[Medline]
5. Sharp JJ, Mobraaten LE. To save or not to save: the role of repositories in a period of rapidly expanding development of genetically engineered strains of mice. In: Houdebine LM (ed.), Transgenic Animals: Generation and Use. Netherlands: Harwood Academic Publishers; 1997: 525–532.
6. Whittingham DG, Leibo SP, Mazur P. Survival of mouse embryos frozen to -196°C and -269°C. Science 1972; 178:411–414.[Abstract/Free Full Text]
7. Mazur P. Freezing of living cells: mechanisms and implications. Am J Physiol 1984; 247:C125-C142.
8. Songsasen N, Betteridge KJ, Leibo SP. Birth of live mice resulting from oocytes fertilized in vitro with cryopreserved spermatozoa. Biol Reprod 1997; 56:143–152.[Abstract]
9. Tanphaichitr N, Zheng YS, Kates M, Abdullah N, Chan A. Cholesterol and phospholipid levels of washed and percoll gradient centrifuged mouse sperm: presence of lipids possessing inhibitory effects on sperm motility. Mol Reprod Dev 1996; 43:187–195.[CrossRef][Medline]
10. Tada N, Sato M, Yamonoi J, Mizorogi T, Kasai K, Ogawa S. Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol. J Reprod Fertil 1990; 89:511–516.[Abstract/Free Full Text]
11. Yokoyama M, Akiba H, Katsuki M, Nomura T. Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa. Exp Anim 1990; 39:125–128.
12. Mobraaten LE, Champlin AK, Johnston DS, Schroeder AC, Gordon JW. Cryopreservation and in vitro fertilization with mouse sperm. Cryobiology 1991; 28:527–528.
13. Sztein JM, Schmidt PM, Raber J, Rall WF. Cryopreservation of mouse spermatozoa in a glycerol/raffinose solution. Cryobiology 1992; 29:736–737.
14. Nakagata N, Takeshima T. High fertilizing ability of mouse spermatozoa diluted slowly after cryopreservation. Theriogenology 1992; 37:1238–1291.
15. Penfold LM, Moore HDM. A new method for cryopreservation of mouse spermatozoa. J Reprod Fertil 1993; 99:131–134.[Abstract/Free Full Text]
16. Tao J, Du J, Kleinhans FW, Critser ES, Mazur P, Critser JK. The effect of collection temperature, cooling rate and warming rate on chilling injury and cryopreservation of mouse spermatozoa. J Reprod Fertil 1995; 104:231–236.[Abstract/Free Full Text]
17. Songsasen N, Leibo SP. Relationship between survival after cryopreservation and osmotic tolerance of mouse sperm from different strains. Cryobiology 1996; 33:634.
18. Songsasen N, Leibo SP. Cryopreservation of mouse spermatozoa: II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from 3 strains of mice. Cryobiology 1997; 35:240–254.[CrossRef][Medline]
19. Fuller SJ, Whittingham DG. Effect of cooling mouse spermatozoa to 4°C on fertilization and embryonic development. J Reprod Fertil 1996; 108:139–145.[Abstract/Free Full Text]
20. Willoughby CE, Mazur P, Peter AT, Critser JK. Osmotic tolerance limits and properties of murine spermatozoa. Biol Reprod 1996; 55:715–727.[Abstract]
21. Noiles EE, Thompson KA, Storey BT. Water permeability, L_p, of mouse sperm plasma membrane and its activation energy are strongly dependent on interaction of the plasma membrane with the sperm cytoskeleton. Cryobiology 1997; 35:79–92.[CrossRef][Medline]
22. Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK. Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod 1995; 53:985–995.[Abstract]
23. Gilmore JA, Liu J, Peter AT, Critser JK. Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod 1998; 58:28–36.[Abstract/Free Full Text]
24. Kedem O, Katchalsky A. Thermodynamic analysis of the permeability of biological membranes to nonelectrolytes. Biochim Biophys Acta 1958; 27:229–246.[Medline]
25. Levin RL, Cravalho EG, Huggins CE. A membrane model describing the effect of temperature on water conductivity of erythrocyte membranes at subzero temperatures. Cryobiology 1976; 13:419–429.
26. Gilmore JA, Liu J, Gao DY, Critser JK. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum Reprod 1997; 12:112–118.
27. Devireddy RV, Swanlund DJ, Roberts KP, Bischof JC. Water permeability parameters of mouse sperm during freezing in the presence of extracellular ice and glycerol. Cryobiology 1998; 37:416–417.
28. Crowe JH, Hoekstra FA, Crowe LM, Anchordoguy TJ, Drobnis E. Lipid phase transitions measured in intact cells with Fourier transform infared spectroscopy. Cryobiology 1989; 26:76–84.[CrossRef][Medline]
29. Drobnis EZ, Crowe LM, Berger T, Anchordoguy TJ, Overstreet JW, Crowe JH. Cold shock damage is due to lipid phase transitions in cell membranes: a demonstration using sperm as a model. J Exp Zool 1993; 265:432–437.[CrossRef][Medline]
30. Noiles EE, Bailey JL, Storey BT. The temperature dependence in the hydraulic conductivity, Lp, of the mouse sperm plasma membrane shows a discontinuity between 4 and 0°C. Cryobiology 1995; 32:220–238.[CrossRef][Medline]
31. Quinn PJ. Lipid-phase separation model of low-temperature damage to biological membranes. Cryobiology 1985; 22:128–146.[CrossRef][Medline]
32. Storey BT, Noiles EE, Thompson KA. Comparison of glycerol, other polyols, trehalose, and raffinose to provide a defined cryoprotectant medium for mouse cryopreservation. Cryobiology 1998; 37:46–58.[CrossRef][Medline]

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