Subzero Water Permeability Parameters of Mouse Spermatozoa in the Presence of Extracellular Ice and Cryoprotective Agents1

Abstract

Optimization of techniques for cryopreservation of mammalian sperm is limited by a lack of knowledge regarding water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPAs). Cryomicroscopy cannot be used to measure dehydration during freezing in mammalian sperm because they are highly nonspherical and their small dimensions are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of ICR mouse epididymal sperm cell suspensions was obtained at cooling rates of 5 and 20 degrees C/min in the presence of extracellular ice and CPAs. Using previously published data, the mouse sperm cell was modeled as a cylinder (122-microm long, radius 0.46 microm) with an osmotically inactive cell volume (V(b)) of 0.61V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best-fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The "combined best-fit" membrane permeability parameters at 5 and 20 degrees C/min for mouse sperm cells in solution are as follows: in D-PBS: L(pg) = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp) = 94.1 kJ/mole (22.5 kcal/mole) (R(2) = 0.94); in "low" CPA media (consisting of 1% glycerol, 6% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 1.7 x 10(-15) m(3)/Ns (0.01 microm/min-atm) and E(Lp)[cpa] = 122.2 kJ/mole (29.2 kcal/mole) (R(2) = 0.98); and in "high" CPA media (consisting of 4% glycerol, 16% raffinose, and 15% egg yolk in D-PBS): L(pg)[cpa] = 0.68 x 10(-15) m(3)/Ns (0.004 microm/min-atm) and E(Lp)[cpa] = 63.6 kJ/mole (15.2 kcal/mole) (R(2) = 0.99). These parameters are significantly different than previously published parameters for mammalian sperm obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in mouse sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPAs. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates, 10-40 degrees C/min, and the numerically predicted optimal cooling rates, greater than 5000 degrees C/min, obtained using suprazero mouse sperm permeability parameters that do not account for the presence of extracellular ice. As an independent test of this prediction, the percentages of viable and motile sperm cells were obtained after freezing at two different cooling rates ("slow" or 5 degrees C/min; "fast," or 20 degrees C/min) in both the low and high CPA media. The greatest sperm motility and viability was found with the low CPA media under fast (20 degrees C/min) cooling conditions.

Effect of glycerol and cholesterol-loaded cyclodextrin on freezing-induced water loss in bovine spermatozoa

“…experimental data can be reconciled (Devireddy et al 1999, Curry 2000. The best-fit parameters obtained in this study with the DSC water transport data (shown in Table 1) during freezing of bovine spermatozoa confirm that this is indeed the case; L pg ¼ 0.02-0.036 mm/min-atm and E Lp ¼ 26.4-42.1 kcal/mol.…”

“…A dissimilarity between the suprazero and subzero water transport parameters was also found for mouse (Devireddy et al 1999), human (Devireddy et al 2000), horse (Devireddy et al 2002a,b), dog (Thirumala et al 2003) and boar sperm cells. This discrepancy between the membrane permeabilities may be associated with possible changes in the sperm cell plasma membrane during suprazero cooling, including a lipid-phase transition of 0 -4 8C (Noiles et al 1995) and/or cold-shock damage or 'chilling' injury during cooling (Blok et al 1976, Watson 1981, Steponkus 1984.…”

“…We chose to study membrane transport in the presence of glycerol, since bovine sperm is routinely cryopreserved in the presence of 4 -8% v/v glycerol (Rodriguez et al 1975, Robbins et al 1976; for a review of various bovine sperm-preservation media, see Vishwanath & Shannon 2000). Stepwise addition of CPAs was performed at 25 8C to minimize the osmotic injury and to lessen the volumetric excursions of bovine spermatozoa during the CPA-loading process (Liu & Foote 1998, Devireddy et al 1999. At room temperature (25 8C), a stock of 1.4 M CPA was added to the sperm cells in five equal volume steps at 5-min intervals such that the final concentration of the CPA was 0.7 M. The equilibration time and the number of steps were chosen on the basis of equations developed by Kedem and Katchalsky (1958), and utilizing the suprazero (obtained in the absence of extracellular ice) membrane permeability parameter values (L p , 0.7 m m/min-atm and E a , 10 kcal/mol) reported in the literature for bovine sperm , Chaveiro et al 2004.…”

“…At cooling rates over 60 8C/min, the amount of water trapped inside the tissue cells increases rapidly with increasing cooling rate. A more detailed analysis of the water transport simulations was also performed, as described in earlier studies (Devireddy et al 1999, Thirumala et al 2003, to determine the optimal rate of freezing bovine sperm in the three freezing media investigated. Briefly, we found that the predicted optimal rates of freezing obtained by analyzing the water transport simulations are 43 8C/min, 57 8C/min and 62 8C/min for bovine sperm frozen in the absence of CPAs, in the presence of glycerol and in the presence of CLC respectively.…”

“…However, a novel method using a differential scanning calorimeter (DSC) has enabled the measurement of the water transport response during freezing of spermatozoa of several species, including mouse (Devireddy et al 1999), man (Devireddy et al 2000), horse (Devireddy et al 2002a,b), dog (Thirumala et al 2003), Pacific oyster (He et al 2004), boar , green swordtail and southern platyfish (Pinisetty et al 2005). This DSC technique was used in this study to measure the membrane permeability parameters of bovine spermatozoa at a cooling rate of 20 8C/min in three different media: 1. in the absence of any cryoprotective agents (CPAs); 2. in the presence of 0.7 M glycerol; 3. in the presence of 1.5 mg/ml CLC and 0.7 M glycerol.…”