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41. Membrane permeability of rhesus monkey oocytes to water, propylene glycol, ethylene glycol and dimethylsulfoxide
Abstracts / Cryobiology 57 (2008) 315–340 blastocysts is high, and suggested that channel processes are responsible for the high permeability. In this study, we examined the pathway for the movement of water and cryoprotectants in bovine oocytes and embryos at various developmental stages. Bovine oocytes and embryos were exposed to PB1 medium containing 0.44 M sucrose, 10% glycerol (Gly), 8.5% ethylene glycol (EG), 9.5% Me2SO or 10% propylene glycol (PG) at 15 and 25 °C for 10 min, to calculate their permeabilities to water (LP) and cryoprotectants (PS) and the activation energy (Ea) for the permeabilities. The LP in the sucrose solution and PGly and PEG of oocytes and 16-cell embryos were low and the Ea for the permeabilities was high, whereas the LP and PS of morulae and blastocysts were high and the Ea for the permeabilities was low. Thus, in oocytes and 16-cell embryos, water, Gly and EG must move across the plasma membrane predominantly via simple diffusion, whereas, in morulae and blastocysts, these molecules must move predominantly via channel processes. On the other hand, PS values for Me2SO and PG did not differ significantly among oocytes and embryos at various stages, and the Ea for the permeabilities was high. Thus, Me2SO and PG would permeate oocytes and embryos predominantly by simple diffusion. Therefore, in bovine morulae and blastocysts, channel processes would be responsible for the high permeability to water and some cryoprotectants as in mouse embryos. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.039
39. Vitrification of immature ovine oocytes with Cryoloop: Effect on in vitro fertilization rates. Adel R. Moawad, Dasari Amarnath, Inchul Choi, Pat Fisher, Jie Zhu, Keith H.S. Campbell, University of Nottingham, Loughborough, United Kingdom Successful cryopreservation of mammalian oocytes is crucial for gene banking, species preservation and assisted reproductive technologies. The aims of this work were (1) to evaluate meiotic maturation of immature ovine oocytes exposed to different vitrification solutions, (2) to study the effect of CryoLoop vitrification on in vitro fertilization of immature ovine oocytes. Experiment 1: cumulus oocyte complexes (COCs) obtained from slaughtered sheep were exposed to VSI or VSII or VSIII [Yamada et al., Anim. Reprod. Sci. 99 (2007) 384–388]. After 24 h in vitro maturation [Lee, Campbell, Biol. Reprod. 74 (2006) 691–698], oocytes were stained with orcin to evaluate maturation rates. Experiment 2: COCs were randomly divided into three groups (1) untreated (control), (2) exposed to vitrification solutions without freezing (toxicity), (3) vitrified by CryoLoop [Moawad, Campbell, Reprod. Fertil. Dev. 20 (2008) 122]. Oocytes were fertilized in vitro using 2 106 sperm/ml. After 18 h of gamete co-incubation, fertilization rates were determined using Hoechst 33342. Survival of oocytes was 81.9, 75.3 and 81.8 for VSI, VSII and VIII, respectively. Maturation rate was the lowest (P < 0.001) for oocytes exposed to VSII (16.1%) than VSI, VSIII and control oocytes (50.0%, 45.1%, and 61.4%, respectively). After CryoLoop vitrification (82.6% 161/195) of oocytes were recovered. No differences in percentages of fertilized oocytes in vitrified, toxicity and control groups (54.0%, 75.0% and 74.1%, respectively). CryoLoop vitrification resulted in significantly higher (P < 0.001) polyspermy (17.0%) than control (3.5%). Results suggest that exposure of immature ovine oocytes to higher concentrations of cryoprotectant (25% EG + 25% Me2SO) has an adverse effect on meiotic maturation. Immature ovine oocytes vitrified with CryoLoop can be fertilized in vitro with higher fertilization rates. However, further studies are needed to evaluate the developmental competence of vitrified immature ovine oocytes. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.040
40. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling. Peter Mazur, Shinsuke Seki, University of Tennessee, Knoxville, TN, USA The formation of ice within cells (IIF) is generally lethal, and successful cryopreservation requires that it be avoided. In most cases IIF occurs during the too rapid cooling of cells to temperatures below 40 °C, but in some cases it is manifested not during cooling but during warming. In such cases, the cell water has vitrified during cooling, and IIF becomes manifest during warming as a consequence of devitrification followed by recrystallization (the conversion of small ice crystals into large). A recent paper from our laboratory [Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to 25 °C, holding them 10 min, rapidly cooling them to 70 °C, and warming them slowly until thawed. IIF, as evidenced by blackening of the cells, became detectable at 56 °C and was complete by 46 °C. The present study differed in that the oocytes were warmed rapidly from 70 °C to temperatures between 65 °C and 50 °C and held for 2 to 60 minutes. The permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (Ea) for the blackening process. The value of Ea is 27.5 kcal/mole, more than double typical values. This translates to about a quintupling of the blackening rate for every 5 °C rise in temper-
323
ature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. The higher the warming rate, the higher the temperature at which a given degree of blackening occurred. These findings have significant implications both for cryobiology and cryo-electron microscopy. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.041
41. Membrane permeability of rhesus monkey oocytes to water, propylene glycol, ethylene glycol and dimethylsulfoxide. Jens O.M. Karlsson a, Abdelmoneim I. Younis b,c, Anthony Chan c, Kenneth G. Gould c, Ali Eroglu d, a Villanova University, Villanova, PA, USA, b Mercer University School of Medicine, Macon, GA, USA, c Yerkes, National Primate Research Center, Atlanta, GA, USA, d Medical College of Georgia, Augusta, GA, USA Oocytes of non-human primates such as rhesus monkeys (Macaca mulatta) have proven extremely valuable as a research model; however, they are very costly to obtain, and seasonal availability restricts their use. A successful cryopreservation technique would overcome these limitations and also help the conservation of endangered primate species. Furthermore, successful cryopreservation of non-human primate oocytes would provide a stepping stone for human oocyte cryopreservation. In the present study, we investigated the membrane permeability of rhesus monkey oocytes to water and three widely used cryoprotectants at room temperature (23.5 °C). We report the first measurement of permeability to propylene glycol (PROH) in rhesus oocytes, and also present permeability data for ethylene glycol (EG) and dimethylsulfoxide (Me2SO). Our results indicate that rhesus oocytes from all meiotic stages are more permeable to PROH than to EG and Me2SO. On the other hand, the hydraulic conductivity was higher in the presence of Me2SO than in the presence of EG or PROH, or in the absence of cryoprotectants altogether. Overall, mature oocytes tended to be more permeable to the cryoprotectants and less permeable to water than were immature oocytes. The biophysical parameters measured in this study were used in computer-aided design of optimal cryoprotectant loading and dilution protocols, providing a basis for development of a successful cryopreservation technique for primate oocytes. (Conflicts of interest: None declared. Source of funding: National Institute of Child Health and Human Development Grant Number R01HD049537, awarded to A.E.) doi:10.1016/j.cryobiol.2008.10.042
42. Improved recovery of human oocytes following non-linear cooling. John Morris a, Martyn Blaney b, a Asymptote Ltd, Cambridge, United Kingdom, b Bourn Hall Clinic, Cambridge, United Kingdom Cryopreservation of spermatozoa and embryos is widely used in IVF, by comparison the role of oocyte cryopreservation in assisted reproduction is limited. There are many technical problems associated with cryopreservation of human oocytes either by slow cooling or by vitrification which have limited clinical progress. Many of the changes in physical properties which occur in an aqueous cryoprotectant following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of solutes include ionic species and cryoprotectants, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature. In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a non-linear manner with temperature. Conventional approaches to cryopreservation impose a linear change of temperature with time whilst the stresses that cells are encountering are all non-linear with time. We have demonstrated that improved methods of cryopreservation may be developed for a number of celltypes by specifically manipulating the manner in which cells experience physical changes rather than imposing a linear temperature reduction. In this study we examined human oocytes suspended in a standard cryoprotectant, frozen using a non-linear profile and the effects on post thaw survival and function assessed in comparison with conventional, linear techniques. The only variable was the temperature profile from 7 °C (ice nucleation) to 31 °C (initiation of rapid cooling), the elapsed time of the two treatments was identical. The recovery following non linear cooling was significantly improved (83%) compared with linear cooling (62%). (Conflicts of interest: J. Morris is a shareholder in Asymptote Ltd. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.043
43. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice. Shinsuke Seki, Peter Mazur, University of Tennessee, Knoxville, TN, USA
39. Vitrification of immature ovine oocytes with Cryoloop: Effect on in vitro fertilization rates. Adel R. Moawad, Dasari Amarnath, Inchul Choi, Pat Fisher, Jie Zhu, Keith H.S. Campbell, University of Nottingham, Loughborough, United Kingdom Successful cryopreservation of mammalian oocytes is crucial for gene banking, species preservation and assisted reproductive technologies. The aims of this work were (1) to evaluate meiotic maturation of immature ovine oocytes exposed to different vitrification solutions, (2) to study the effect of CryoLoop vitrification on in vitro fertilization of immature ovine oocytes. Experiment 1: cumulus oocyte complexes (COCs) obtained from slaughtered sheep were exposed to VSI or VSII or VSIII [Yamada et al., Anim. Reprod. Sci. 99 (2007) 384–388]. After 24 h in vitro maturation [Lee, Campbell, Biol. Reprod. 74 (2006) 691–698], oocytes were stained with orcin to evaluate maturation rates. Experiment 2: COCs were randomly divided into three groups (1) untreated (control), (2) exposed to vitrification solutions without freezing (toxicity), (3) vitrified by CryoLoop [Moawad, Campbell, Reprod. Fertil. Dev. 20 (2008) 122]. Oocytes were fertilized in vitro using 2 106 sperm/ml. After 18 h of gamete co-incubation, fertilization rates were determined using Hoechst 33342. Survival of oocytes was 81.9, 75.3 and 81.8 for VSI, VSII and VIII, respectively. Maturation rate was the lowest (P < 0.001) for oocytes exposed to VSII (16.1%) than VSI, VSIII and control oocytes (50.0%, 45.1%, and 61.4%, respectively). After CryoLoop vitrification (82.6% 161/195) of oocytes were recovered. No differences in percentages of fertilized oocytes in vitrified, toxicity and control groups (54.0%, 75.0% and 74.1%, respectively). CryoLoop vitrification resulted in significantly higher (P < 0.001) polyspermy (17.0%) than control (3.5%). Results suggest that exposure of immature ovine oocytes to higher concentrations of cryoprotectant (25% EG + 25% Me2SO) has an adverse effect on meiotic maturation. Immature ovine oocytes vitrified with CryoLoop can be fertilized in vitro with higher fertilization rates. However, further studies are needed to evaluate the developmental competence of vitrified immature ovine oocytes. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.040
40. Kinetics and activation energy of recrystallization of intracellular ice in mouse oocytes subjected to interrupted rapid cooling. Peter Mazur, Shinsuke Seki, University of Tennessee, Knoxville, TN, USA The formation of ice within cells (IIF) is generally lethal, and successful cryopreservation requires that it be avoided. In most cases IIF occurs during the too rapid cooling of cells to temperatures below 40 °C, but in some cases it is manifested not during cooling but during warming. In such cases, the cell water has vitrified during cooling, and IIF becomes manifest during warming as a consequence of devitrification followed by recrystallization (the conversion of small ice crystals into large). A recent paper from our laboratory [Cryobiology 55 (2007) 158–166] dealt with one such case in mouse oocytes. It involved rapidly cooling the oocytes to 25 °C, holding them 10 min, rapidly cooling them to 70 °C, and warming them slowly until thawed. IIF, as evidenced by blackening of the cells, became detectable at 56 °C and was complete by 46 °C. The present study differed in that the oocytes were warmed rapidly from 70 °C to temperatures between 65 °C and 50 °C and held for 2 to 60 minutes. The permitted us to determine the rate of blackening as function of temperature. That in turn allowed us to calculate the activation energy (Ea) for the blackening process. The value of Ea is 27.5 kcal/mole, more than double typical values. This translates to about a quintupling of the blackening rate for every 5 °C rise in temper-
323
ature. These data then allowed us to compute the degree of blackening as a function of temperature for oocytes warmed at rates ranging from 10 to 10,000 °C/min. The higher the warming rate, the higher the temperature at which a given degree of blackening occurred. These findings have significant implications both for cryobiology and cryo-electron microscopy. (Conflicts of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.041
41. Membrane permeability of rhesus monkey oocytes to water, propylene glycol, ethylene glycol and dimethylsulfoxide. Jens O.M. Karlsson a, Abdelmoneim I. Younis b,c, Anthony Chan c, Kenneth G. Gould c, Ali Eroglu d, a Villanova University, Villanova, PA, USA, b Mercer University School of Medicine, Macon, GA, USA, c Yerkes, National Primate Research Center, Atlanta, GA, USA, d Medical College of Georgia, Augusta, GA, USA Oocytes of non-human primates such as rhesus monkeys (Macaca mulatta) have proven extremely valuable as a research model; however, they are very costly to obtain, and seasonal availability restricts their use. A successful cryopreservation technique would overcome these limitations and also help the conservation of endangered primate species. Furthermore, successful cryopreservation of non-human primate oocytes would provide a stepping stone for human oocyte cryopreservation. In the present study, we investigated the membrane permeability of rhesus monkey oocytes to water and three widely used cryoprotectants at room temperature (23.5 °C). We report the first measurement of permeability to propylene glycol (PROH) in rhesus oocytes, and also present permeability data for ethylene glycol (EG) and dimethylsulfoxide (Me2SO). Our results indicate that rhesus oocytes from all meiotic stages are more permeable to PROH than to EG and Me2SO. On the other hand, the hydraulic conductivity was higher in the presence of Me2SO than in the presence of EG or PROH, or in the absence of cryoprotectants altogether. Overall, mature oocytes tended to be more permeable to the cryoprotectants and less permeable to water than were immature oocytes. The biophysical parameters measured in this study were used in computer-aided design of optimal cryoprotectant loading and dilution protocols, providing a basis for development of a successful cryopreservation technique for primate oocytes. (Conflicts of interest: None declared. Source of funding: National Institute of Child Health and Human Development Grant Number R01HD049537, awarded to A.E.) doi:10.1016/j.cryobiol.2008.10.042
42. Improved recovery of human oocytes following non-linear cooling. John Morris a, Martyn Blaney b, a Asymptote Ltd, Cambridge, United Kingdom, b Bourn Hall Clinic, Cambridge, United Kingdom Cryopreservation of spermatozoa and embryos is widely used in IVF, by comparison the role of oocyte cryopreservation in assisted reproduction is limited. There are many technical problems associated with cryopreservation of human oocytes either by slow cooling or by vitrification which have limited clinical progress. Many of the changes in physical properties which occur in an aqueous cryoprotectant following ice nucleation are not linear with temperature. Parameters such as the ice fraction, concentration of solutes include ionic species and cryoprotectants, osmolality, pH, viscosity and gas solubility, all vary in a non-linear manner with temperature. In addition, the biophysical characteristics of cells which determine the response to freezing, for example the cellular permeability to water, also change in a non-linear manner with temperature. Conventional approaches to cryopreservation impose a linear change of temperature with time whilst the stresses that cells are encountering are all non-linear with time. We have demonstrated that improved methods of cryopreservation may be developed for a number of celltypes by specifically manipulating the manner in which cells experience physical changes rather than imposing a linear temperature reduction. In this study we examined human oocytes suspended in a standard cryoprotectant, frozen using a non-linear profile and the effects on post thaw survival and function assessed in comparison with conventional, linear techniques. The only variable was the temperature profile from 7 °C (ice nucleation) to 31 °C (initiation of rapid cooling), the elapsed time of the two treatments was identical. The recovery following non linear cooling was significantly improved (83%) compared with linear cooling (62%). (Conflicts of interest: J. Morris is a shareholder in Asymptote Ltd. Source of funding: None declared.) doi:10.1016/j.cryobiol.2008.10.043
43. Effect of warming rate on the survival of vitrified mouse oocytes and on the recrystallization of intracellular ice. Shinsuke Seki, Peter Mazur, University of Tennessee, Knoxville, TN, USA