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The prediction of water loss from ram spermatozoa during cryopreservation
562
ABSTRACTS, 25th ANNUAL MEETING
spermatozoa, even a transient membrane reorganization might bring with it some detrimental effects like increased membrane permeability, loss of ions and reduction of membrane pump function. Comparable results were obtained in 1984 by Holt and North (J. Exp. Zool. 230, 473). Differences between their observations on ram spermatozoa and the present observations on bull sperm can be explained in the first place by deviations in the experimental method used. Holt and North added chemical fixatives and cryoprotectants, so artifacts due to these substances can not be ruled out. Second, it seems likely that speciesspecific variation in membrane phospholipid composition causes different phase transition behavior and is responsible for the observed differences in membrane changes.
of Biomedical Engineering, Cambridge, Massachusetts),
The biophysical parameters that govern water transport in a cell are hydraulic permeability and the cell’s activation energy. These parameters were measured in our laboratory for unfertilized and fertilized mouse ova. Metaphase II oocytes and sperm were obtained from B6D2F, mice. For experiments done with unfertilized ova, oocytes were freed from their cumulus masses by treating them with hyaluronidase. For fertilized experiments, sperm was added to culture dishes containing cumulus masses; the dishes were placed in a 5% CO, incubator for 10 hr in which time fertilization took place. The volumetric response of ova was observed during freezing at a constant cooling rate of 2”C/min on isothermal cryostage. Control of the temperature of the cryostage was accomplished by a com154. The Prediction of Water Loss from Ram Spermaputer-based system. The response of ova were retozoa during Cryopreservaiion. P. F. WATSON corded on videotape and subsequently digitized, and a AND ANNE E. DUNCAN (Department of Vetervolume/temperature history of the ova during freezing inary Basic Sciences, Royal Veterinary Colwas generated by electronically measuring the prolege, Royal College Street, London NW1 OTU, jected cross-sectional area of the digitized images. The United Kingdom). hydraulic permeability and the activation energy for each experiment were determined simultaneously by Model curves of cell dehydration during cryopresfitting the volume/temperature data with a theoretical ervation have been established for a number of cell volume/temperature history predicted by the kinetics types with moderate to good agreement with empirical of water transport. A multiple-variable, nonlinear reresults. The necessary parameters measured are cell gression analysis was used to lit the data. The hydrauwater content, surface area, hydraulic conductivity, lic permeability at 0°C of unfertilized ova was deterand its temperature dependence. Our own attempts to mined to be 0.044 pm/min atm * a standard deviation estimate values for the parameters of ram spermatozoa of 0.008 pm/min atm. Permeability for fertilized ova have been hampered by the peculiar shape and rewas found to be 0.079 f 0.028 pm/min atm. Activation gional specialization of structure and function of these energies were found to be 13.30 ? 2.48 kcal/mol for cells. Methods have been devised to yield tentative unfertilized and 19.32 * 4.44 kcal/mol for fertilized values, and water loss curves during freezing at differova. A probabilistic computer model was developed to ent cooling rates have been constructed. Agreement simulate water transport from ova during freezing uswith empirical data was poor, but it is not certain ing the measured distributions of biophysical paramewhether this is due to inaccurate estimation of the paters. From this model the probable distribution of cell rameter values or to incorrect assumptions inherent in volume during freezing was determined, and the probthe equations used to estimate water loss. Spermatoability of intracellular nucleation of the ova was deterzoa have a very low water content compared with mined using the amount of supercooling of the cytoother cell types, and have a rigid internal structure plasm as the criterion for nucleation, composed of dense nucleus and axoneme. They also have membrane bounded organelles confined to cer156. Thermotropic Response of Human Oocyte durtain regions (e.g., mitochondria and acrosome). These ing Measurement of Hydraulic Conductivity features may well complicate the process of water dif(LP): A Comparison with Mouse Oocytes. J. fusion and cell shrinkage, and render the equations too HUNTER, A. BERNARD, B. J. FULLER, J. J. simplistic. Complementary techniques are being used MCGRATH, C. IFFLAND, W. REID, AND R. W. where possible to ensure accurate parameter estimaSHAW (Department of Obstetrics & Gynaecoltion, in order to establish the reasons for the discrepogy, Royal Free Hospital, London, United ancy between theoretical and empirical results. Kingdom). 155. Experimental
Distribution of Permeability Parameters of Mouse Ova and Their Behavior During Freezing. M. R. ORRICO, M. TONER,
D. R. ARMANT,AND E. G. CRAVALHO(Massachusetts Institute of Technology, Department
Although human embryo freezing is now common practice in many IVF units, routine low-temperature storage of human occytes, in our view, has not yet been achieved. If oocytes could be stored for later fertilization and embryo transfer, many of the ethical
ABSTRACTS, 25th ANNUAL MEETING
spermatozoa, even a transient membrane reorganization might bring with it some detrimental effects like increased membrane permeability, loss of ions and reduction of membrane pump function. Comparable results were obtained in 1984 by Holt and North (J. Exp. Zool. 230, 473). Differences between their observations on ram spermatozoa and the present observations on bull sperm can be explained in the first place by deviations in the experimental method used. Holt and North added chemical fixatives and cryoprotectants, so artifacts due to these substances can not be ruled out. Second, it seems likely that speciesspecific variation in membrane phospholipid composition causes different phase transition behavior and is responsible for the observed differences in membrane changes.
of Biomedical Engineering, Cambridge, Massachusetts),
The biophysical parameters that govern water transport in a cell are hydraulic permeability and the cell’s activation energy. These parameters were measured in our laboratory for unfertilized and fertilized mouse ova. Metaphase II oocytes and sperm were obtained from B6D2F, mice. For experiments done with unfertilized ova, oocytes were freed from their cumulus masses by treating them with hyaluronidase. For fertilized experiments, sperm was added to culture dishes containing cumulus masses; the dishes were placed in a 5% CO, incubator for 10 hr in which time fertilization took place. The volumetric response of ova was observed during freezing at a constant cooling rate of 2”C/min on isothermal cryostage. Control of the temperature of the cryostage was accomplished by a com154. The Prediction of Water Loss from Ram Spermaputer-based system. The response of ova were retozoa during Cryopreservaiion. P. F. WATSON corded on videotape and subsequently digitized, and a AND ANNE E. DUNCAN (Department of Vetervolume/temperature history of the ova during freezing inary Basic Sciences, Royal Veterinary Colwas generated by electronically measuring the prolege, Royal College Street, London NW1 OTU, jected cross-sectional area of the digitized images. The United Kingdom). hydraulic permeability and the activation energy for each experiment were determined simultaneously by Model curves of cell dehydration during cryopresfitting the volume/temperature data with a theoretical ervation have been established for a number of cell volume/temperature history predicted by the kinetics types with moderate to good agreement with empirical of water transport. A multiple-variable, nonlinear reresults. The necessary parameters measured are cell gression analysis was used to lit the data. The hydrauwater content, surface area, hydraulic conductivity, lic permeability at 0°C of unfertilized ova was deterand its temperature dependence. Our own attempts to mined to be 0.044 pm/min atm * a standard deviation estimate values for the parameters of ram spermatozoa of 0.008 pm/min atm. Permeability for fertilized ova have been hampered by the peculiar shape and rewas found to be 0.079 f 0.028 pm/min atm. Activation gional specialization of structure and function of these energies were found to be 13.30 ? 2.48 kcal/mol for cells. Methods have been devised to yield tentative unfertilized and 19.32 * 4.44 kcal/mol for fertilized values, and water loss curves during freezing at differova. A probabilistic computer model was developed to ent cooling rates have been constructed. Agreement simulate water transport from ova during freezing uswith empirical data was poor, but it is not certain ing the measured distributions of biophysical paramewhether this is due to inaccurate estimation of the paters. From this model the probable distribution of cell rameter values or to incorrect assumptions inherent in volume during freezing was determined, and the probthe equations used to estimate water loss. Spermatoability of intracellular nucleation of the ova was deterzoa have a very low water content compared with mined using the amount of supercooling of the cytoother cell types, and have a rigid internal structure plasm as the criterion for nucleation, composed of dense nucleus and axoneme. They also have membrane bounded organelles confined to cer156. Thermotropic Response of Human Oocyte durtain regions (e.g., mitochondria and acrosome). These ing Measurement of Hydraulic Conductivity features may well complicate the process of water dif(LP): A Comparison with Mouse Oocytes. J. fusion and cell shrinkage, and render the equations too HUNTER, A. BERNARD, B. J. FULLER, J. J. simplistic. Complementary techniques are being used MCGRATH, C. IFFLAND, W. REID, AND R. W. where possible to ensure accurate parameter estimaSHAW (Department of Obstetrics & Gynaecoltion, in order to establish the reasons for the discrepogy, Royal Free Hospital, London, United ancy between theoretical and empirical results. Kingdom). 155. Experimental
Distribution of Permeability Parameters of Mouse Ova and Their Behavior During Freezing. M. R. ORRICO, M. TONER,
D. R. ARMANT,AND E. G. CRAVALHO(Massachusetts Institute of Technology, Department
Although human embryo freezing is now common practice in many IVF units, routine low-temperature storage of human occytes, in our view, has not yet been achieved. If oocytes could be stored for later fertilization and embryo transfer, many of the ethical