- Email: [email protected]
Life on pause: epigenetic mechanisms underlie metabolic stasis in cold-adapted animals
Abstracts / Cryobiology 73 (2016) 399e443
well. Here, various aspects of these recent advances are outlined and discussed, along with their relevance to cryobiology. For instance, it has been shown that various solute molecules can have significant and unpredictable impacts on ice nucleation temperatures, with both enhancements and suppressions of ice nucleation observed. These effects have the potential to influence outcomes of cryopreservation procedures. Similarly, progress has been made in understanding why certain minerals and biological entities nucleate ice efficiently. This knowledge may allow us to understand and control ice nucleation in cryobiological systems and thereby improve outcomes. Source of funding: European Research Council 632272 IceControl and 648661 MarineIce, National Environmental Research Council NE/I019057/ 1, Asymptote Ltd. S111 PROPERTIES OF SUPERCOOLING-PROMOTING POLYPHENOLS S. Fujikawa 1, *, C. Kuwabara 2, K. Arakawa 2, T. Koyama 3, T. Inada 3. 1 University, Research Faculty and Graduate School of Agriculture, Sapporo, Japan; 2 Hokkaido University, Research Faculty and Graduate School of Agriculture, Sapporo, Japan; 3 National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan * Corresponding author.
Freezing of water is controlled by ice nucleation substances, antifreezing (ice-binding) proteins or glycoproteins, and supercooling-promoting (antiice nucleation) substances. Since supercooling-promoting capability in a few kinds of flavonol glycosides and hydrolysable tannins were found in special cells of trees that adapt by deep supercooling near homogeneous ice nucleation temperature, many related polyphenols have also been recognized as supercooling-promoting substances. Although until now many compounds have been recognized as supercooling-promoting substances, their common properties are obscure. In this study, common properties of supercooling-promoting substances, especially polyphenols, were examined. The supercooling-promoting polyphenols had the ability to reduce sublimation of ice, although they did not affect ice crystal growth. The volume of solutions as well as the presence or absence of vibration affected supercooling capability. The polyphenols promoted supercooling in solutions containing ice nucleators, whereas in the absence of nucleators the polyphenols had no effect on homogeneous ice nucleation. The supercooling capability of individual polyphenols changed largely depending on solutions containing different ice nucleators. Due to such characteristics of the interaction between supercooling-promoting substances and ice nucleators, when any favorite supercooling-promoting substance was applied to solutions such as incubation medium for supercooling preservation of biological materials, the effects changed depending on medium, probably due to difference of contents in the medium. Emulsion freezing assay on interaction of supercooling-promoting substances with other substances, such as sugars and ions, showed that some combination of these substances reduced the homogeneous ice nucleation temperature. Such combinations also allowed supercooling of 1 ml MQ-water under static condition at - 20 C for more than 20 days. Furthermore, similar combination allowed supercooling of 1 ml MQ-water under strong vibration at - 20 C for, at least, one day. S112 INHIBITION OF ICE GROWTH BY A SUPRAMOLECULAR ASSEMBLY R. Drori 1, *, C. Li 1, C. Hu 1, P. Raiteri 2, A. Rohl 2, M. Ward 1, B. Kahr 1. 1 New York University, Department of Chemistry and Molecular Design Institute, New York, New York, United States; 2 Curtin University, Nanochemistry Research Institute, Curtin Institute for Computation and Department of Chemistry, Perth, Western Australia, Australia * Corresponding author.
Control of ice growth can be achieved by using naturally occurring antifreeze proteins, which bind irreversibly to ice and limit its growth. To date, there are no synthetic and effective ice growth inhibitors available. An inexpensive compound was found to inhibit growth and recrystallization
429
of ice at millimolar concentrations; its activity is comparable to highly evolved antifreeze glycoproteins. This compound prevents ice crystals from growing in the a-direction, and bipyramidal needles are obtained. Absorbance measurements indicate that dimerization of this compound occurs in aqueous solutions at concentrations at which it inhibits ice. According to aggregation theory and free energy calculations with classical molecular-dynamics (MD) methods, molecules of this compound stack to form aggregates, in which distinct reactive groups are regularly disposed on the side of the self-associated chains. Layers of structured waters were found by crystallography in aggregated assemblies of this compound, a finding that corresponds to waters found on the ice-binding sites of antifreeze proteins. All of these features suggest that this compound is able to mimic antifreeze proteins by forming a supramolecular antifreeze moiety. Source of funding: This work was supported by the NYU MRSEC Program of the National Science Foundation (NSF) under Award Number DMR1420073, and the Australian Research Council for funding (FT130100463 and DP140101776). The Pawsey Supercomputer Centre and the National Computational Infrastructure, with funding from the Australian Government and the Western Australian Government, are also gratefully acknowledged for the provision of computer time. S113 IMPORTANCE OF THE MITOCHONDRIAL UPR IN AGING R. Tanguay*, G. Morrow. Laval University, Quebec, Canada * Corresponding author.
Aging is characterized by the accumulation of molecular damage that leads to organismal decline and the onset of age-associated disease. At the cellular level, aging is associated with the accumulation of dysfunctional mitochondria. Since these organelles are involved in many important cellular processes, different mechanisms exist to maintain their integrity: reactive oxygen species (ROS) scavenging, mitochondrial unfolding protein response (UPRMT), and mitophagy. While the later aims at clearing defective mitochondria, the UPRMT triggers the expression of a set of proteins aimed at re-establishing mitochondrial homeostasis. The induction of mitochondrial chaperones expression, particularly of Hsp60 and Hsp70, is a hallmark of the UPRMT pathway. In Drosophila melanogaster, Hsp22 is also localized in mitochondria and takes part in the pathway. In addition to being preferentially up-regulated during aging and in oxidative stress conditions, Hsp22 increases lifespan and resistance to stress upon over-expression. Its over-expression also changes the mitoproteomic profile and increases mitochondrial protease activity. Interestingly, Hsp22 co-migrates with components of the electron transport chain upon stress, emphasizing its role in mitochondrial homeostasis. Altogether, the results establish a role of Hsp22 in proteostasis and highlight the role of the UPRMT in preventing the aging process Source of funding: Supported by the Canadian Institutes for Health Research S114 LIFE ON PAUSE: EPIGENETIC MECHANISMS UNDERLIE METABOLIC STASIS IN COLD-ADAPTED ANIMALS K. Storey. Carleton University, Institute of Biochemistry and Department of Biology, Ottawa, Ontario, Canada For many animals, survival of severe environmental stress (e.g., cold, drought, oxygen limitation, food deprivation) is aided by entry into a hypometabolic state. Strong metabolic rate depression, often to only 1-20% of normal resting rate, is the core survival principle of adaptive strategies including hibernation, freeze tolerance, estivation, and anaerobiosis. Global biochemical controls are used to suppress and reprioritize energy use. Recently, we have been exploring the idea that mechanisms previously associated with epigenetic regulation contribute to global suppression of gene expression during all forms of hypometabolism. Coordinated changes in DNA and histone posttranslational modification (e.g., acetylation, methylation, phosphorylation), changes in the activities of histone deacetylases and DNA methyltransferases, and differential expression of microRNA species all appear to underlie the reorganization of cells as they
430
Abstracts / Cryobiology 73 (2016) 399e443
enter hypometabolic states. These modifications are readily reversible and should now be considered as crucial targets to explore and apply to induce stasis in transplantable organs. Epigenetic controls also work hand in hand with the formation of ribonuclear protein bodies in the nucleus and cytoplasm that sequester mRNA transcripts into storage until animals arouse again to normal metabolic function. This research, suggesting a new layer of regulatory control that can contribute to coordinating metabolic rate depression will provide new tools in the arsenal of cryomedical science. For more information visit www.kenstoreylab.com. Source of funding: Supported by NSERC Canada. S115 SURVIVING WINTER: NFATS REGULATE CRYOPROTECTION IN FREEZETOLERANT RANA SYLVATIC R. Al-attar*, K. Storey. Carleton University, Ottawa, Ontario, Canada * Corresponding author.
Cold temperatures can be detrimental to organisms’ health. The freezetolerant Rana sylvatica can survive prolonged periods of whole body freezing (60-75%), which halts a majority of biochemical and physiological processes including breathing before resuming function upon thawing. Freezing prevents oxygen delivery to organs and draws water from the cells to the extracellular matrix for ice crystal formation resulting in anoxia and dehydration. As a result, physiological and biochemical adaptations are crucial for survival during freezing. Some of these adaptations include producing large quantities of glucose (~300 mM), up-regulating stress responsive genes, and down-regulating metabolic rates to 10-20% of normal conditions. Metabolic regulations occur at the transcriptional, translational, and post-translational levels. This study aimed to understand the transcriptional regulation of the nuclear factor of activated T cell 1-4 (NFATs) family of transcription factors in liver and skeletal muscle during the freeze/thaw cycle in Rana sylvatica. Using western blotting and DNA binding assays, we showed that freezing causes differential regulation of NFAT proteins and NFAT DNA binding during 24 h freezing and 8 h thawing in both tissues. The increase in NFATc3 total proteins, nuclear localization and DNA binding activity in the liver may be responsible for inducing the activation of signal transducer and activator of transcription 3 (p-STAT3-S727). Activated STAT3 caused the transcriptional inhibition of the glycogen synthase kinase 3b gene, previously reported to be active in the liver during freezing. In comparison, NFATc3 in skeletal muscle showed no change in protein levels or DNA binding. Interestingly, osteopontin, a downstream target of NFATc3, showed a significant down-regulation in protein expression during 24 h freezing in the skeletal muscle, which may explain the absence of p-STAT3-S727 proteins in this tissue. Together, the data suggests that Rana sylvatica liver, unlike skeletal muscle, actively regulates targets that are involved in glucose metabolism during freezing. Source of funding: This study is funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant (Funding #:6793) awarded to Dr. Kenneth B. Storey. S116 HIBR-MIRS: COLD-SENSITIVE NOVEL MICRORNA IN THE HIBERNATING 13-LINED GROUND SQUIRREL B. Luu 1, *, K. Biggar 1, C. Wu 2, K. Storey 1. 1 Carleton University, Ottawa, Ontario, Canada; 2 Gainesville, Florida, United States * Corresponding author.
As thirteen-lined ground squirrels (Ictidomys tridecemlineatus) enter hibernation, they characteristically switch their primary fuel source from carbohydrates to lipids, depress their basal metabolic rate by 96-e98% to conserve vital energy stores, and significantly drop their core body temperature to 0-e5 C. In recent years, research has demonstrated the importance of microRNA (small noncoding RNA molecules) in coldtolerant animal models, including their dynamic regulation throughout mammalian hibernation. MicroRNA are highly conserved among vertebrates, and function by post-transcriptionally regulating mRNA translation to adapt cells for survival. This study uses small RNA sequencing and
custom bioinformatics pipelines to identify novel species-specific microRNA, and to predict their function. A group of 17 novel microRNA were identified in the ground squirrel (Ictidomys tridecemlineatus), and their relative expression was quantified using real-time PCR in the liver, skeletal muscle, and heart tissues over four experimental conditions that represent the torpor-arousal cycle (euthermic in cold room, early torpor, late torpor, and interbout arousal). These novel microRNAs lacked homology to any other known microRNA in miRBase (the microRNA annotation database). The predicted mRNA targets of these novel microRNA were found to be enriched in biological processes that are known to be regulated during hibernation, such as lipid metabolism, ion-transport ATPases, and various cellular signaling cascades. This study is the first of its kind to identify, quantify, and predict the likely function of species-specific microRNA in a hibernating animal. Source of funding: This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (grant# 6793). K.B.S. holds the Canada Research Chair in Molecular Physiology, B.E.L. holds an NSERC Canada Graduate Scholarship, K.K.B. holds an NSERC Banting postdoctoral fellowship, and CWW holds an NSERC postdoctoral fellowship. S117 BENEFICIAL EFFECTS OF GLUTATHIONE SUPPLEMENT DURING VITRIFICATION OF MOUSE OOCYTES AT THE GERMINAL VESICLE STAGE ON THEIR PREIMPLANTATION DEVELOPMENT FOLLOWING MATURATION AND FERTILIZATION IN VITRO A. Moawad 1, 2, 3, *, S. Tan 1, T. Taketo 1. 1 McGill University, Department of Obstetrics and Gynecology, Canada; 2 Department of Surgery, OriginELLE Fertility Clinic, Montreal Canada; 3 Cairo University, Department of Theriogenology, Giza, Egypt * Corresponding author. McGill University, Department of Obstetrics and Gynecology, Canada.
Cryopreservation of oocytes at the germinal vesicle (GV) stage is an attractive option in ART when immediate IVF or controlled-hormone stimulation is unavailable for a patient; however, the need of IVM diminishes blastocyst development from GV-oocytes and its applications. We previously reported that supplementation of the media with L-carnitine (LC) for vitrification, thawing, and IVM of GV-oocytes improves preimplantation development following IVF in (B6.DBA)F1 and B6 mice. LC has dual biological activities, lipid metabolism and antioxidant, and which activity contributes to the beneficial effects of LC during GV-oocyte cryopreservation remains unknown. The aim of this study was to investigate the effects of glutathione (GSH; a potent antioxidant) supplementation during vitrification and thawing of mouse GV-oocytes on preimplantation development following maturation and fertilization in vitro. Combined effects of GSH and LC during vitrification and IVM were also tested. Our results showed that GSH supplementation in the IVM medium exhibited no inhibitory effects on the cleavage or blastocyst development rate in both mouse strains except that GSH at 1 mM increased the blastocyst development rate in B6 mice. By contrast, GSH supplementation at 1 mM in vitrification and warming media significantly increased blastocyst development rates in both mouse strains. When GSH at 0.5 or 1.0 mM was combined with LC at 1.86 or 3.72 mM in vitrification and warming media, little improvement was found over GSH supplementation alone. However, LC supplementation at 1.86 mM during IVM following vitrification and warming of GV-ooctyes in the media supplemented with both GSH and LC at 0.5 and 3.72 mM, respectively, increased the blastocyst development rate in the (B6.DBA)F1 strain. We conclude that GSH supplementation alone during vitrification and warming of GV-oocytes improves the developmental competence of oocytes, suggesting that the antioxidant activity is important for reducing the damage inflicted by freezing. S118 COLD-SENSITIVE ION CHANNELS ARE INVOLVED IN CHILLING INJURY OF PIG OOCYTES K. Edashige 1, *, Y. Iwahara 1, S. Nariai 1, Y. Nishiya 1, M. Kitayama 1, S. Niimi 1, S. Seki 2, C. Koshimoto 3, K. Matsukawa 1, M. Kasai 1. 1 Kochi
well. Here, various aspects of these recent advances are outlined and discussed, along with their relevance to cryobiology. For instance, it has been shown that various solute molecules can have significant and unpredictable impacts on ice nucleation temperatures, with both enhancements and suppressions of ice nucleation observed. These effects have the potential to influence outcomes of cryopreservation procedures. Similarly, progress has been made in understanding why certain minerals and biological entities nucleate ice efficiently. This knowledge may allow us to understand and control ice nucleation in cryobiological systems and thereby improve outcomes. Source of funding: European Research Council 632272 IceControl and 648661 MarineIce, National Environmental Research Council NE/I019057/ 1, Asymptote Ltd. S111 PROPERTIES OF SUPERCOOLING-PROMOTING POLYPHENOLS S. Fujikawa 1, *, C. Kuwabara 2, K. Arakawa 2, T. Koyama 3, T. Inada 3. 1 University, Research Faculty and Graduate School of Agriculture, Sapporo, Japan; 2 Hokkaido University, Research Faculty and Graduate School of Agriculture, Sapporo, Japan; 3 National Institute of Advanced Industrial Science and Technology (AIST), Ibaraki, Japan * Corresponding author.
Freezing of water is controlled by ice nucleation substances, antifreezing (ice-binding) proteins or glycoproteins, and supercooling-promoting (antiice nucleation) substances. Since supercooling-promoting capability in a few kinds of flavonol glycosides and hydrolysable tannins were found in special cells of trees that adapt by deep supercooling near homogeneous ice nucleation temperature, many related polyphenols have also been recognized as supercooling-promoting substances. Although until now many compounds have been recognized as supercooling-promoting substances, their common properties are obscure. In this study, common properties of supercooling-promoting substances, especially polyphenols, were examined. The supercooling-promoting polyphenols had the ability to reduce sublimation of ice, although they did not affect ice crystal growth. The volume of solutions as well as the presence or absence of vibration affected supercooling capability. The polyphenols promoted supercooling in solutions containing ice nucleators, whereas in the absence of nucleators the polyphenols had no effect on homogeneous ice nucleation. The supercooling capability of individual polyphenols changed largely depending on solutions containing different ice nucleators. Due to such characteristics of the interaction between supercooling-promoting substances and ice nucleators, when any favorite supercooling-promoting substance was applied to solutions such as incubation medium for supercooling preservation of biological materials, the effects changed depending on medium, probably due to difference of contents in the medium. Emulsion freezing assay on interaction of supercooling-promoting substances with other substances, such as sugars and ions, showed that some combination of these substances reduced the homogeneous ice nucleation temperature. Such combinations also allowed supercooling of 1 ml MQ-water under static condition at - 20 C for more than 20 days. Furthermore, similar combination allowed supercooling of 1 ml MQ-water under strong vibration at - 20 C for, at least, one day. S112 INHIBITION OF ICE GROWTH BY A SUPRAMOLECULAR ASSEMBLY R. Drori 1, *, C. Li 1, C. Hu 1, P. Raiteri 2, A. Rohl 2, M. Ward 1, B. Kahr 1. 1 New York University, Department of Chemistry and Molecular Design Institute, New York, New York, United States; 2 Curtin University, Nanochemistry Research Institute, Curtin Institute for Computation and Department of Chemistry, Perth, Western Australia, Australia * Corresponding author.
Control of ice growth can be achieved by using naturally occurring antifreeze proteins, which bind irreversibly to ice and limit its growth. To date, there are no synthetic and effective ice growth inhibitors available. An inexpensive compound was found to inhibit growth and recrystallization
429
of ice at millimolar concentrations; its activity is comparable to highly evolved antifreeze glycoproteins. This compound prevents ice crystals from growing in the a-direction, and bipyramidal needles are obtained. Absorbance measurements indicate that dimerization of this compound occurs in aqueous solutions at concentrations at which it inhibits ice. According to aggregation theory and free energy calculations with classical molecular-dynamics (MD) methods, molecules of this compound stack to form aggregates, in which distinct reactive groups are regularly disposed on the side of the self-associated chains. Layers of structured waters were found by crystallography in aggregated assemblies of this compound, a finding that corresponds to waters found on the ice-binding sites of antifreeze proteins. All of these features suggest that this compound is able to mimic antifreeze proteins by forming a supramolecular antifreeze moiety. Source of funding: This work was supported by the NYU MRSEC Program of the National Science Foundation (NSF) under Award Number DMR1420073, and the Australian Research Council for funding (FT130100463 and DP140101776). The Pawsey Supercomputer Centre and the National Computational Infrastructure, with funding from the Australian Government and the Western Australian Government, are also gratefully acknowledged for the provision of computer time. S113 IMPORTANCE OF THE MITOCHONDRIAL UPR IN AGING R. Tanguay*, G. Morrow. Laval University, Quebec, Canada * Corresponding author.
Aging is characterized by the accumulation of molecular damage that leads to organismal decline and the onset of age-associated disease. At the cellular level, aging is associated with the accumulation of dysfunctional mitochondria. Since these organelles are involved in many important cellular processes, different mechanisms exist to maintain their integrity: reactive oxygen species (ROS) scavenging, mitochondrial unfolding protein response (UPRMT), and mitophagy. While the later aims at clearing defective mitochondria, the UPRMT triggers the expression of a set of proteins aimed at re-establishing mitochondrial homeostasis. The induction of mitochondrial chaperones expression, particularly of Hsp60 and Hsp70, is a hallmark of the UPRMT pathway. In Drosophila melanogaster, Hsp22 is also localized in mitochondria and takes part in the pathway. In addition to being preferentially up-regulated during aging and in oxidative stress conditions, Hsp22 increases lifespan and resistance to stress upon over-expression. Its over-expression also changes the mitoproteomic profile and increases mitochondrial protease activity. Interestingly, Hsp22 co-migrates with components of the electron transport chain upon stress, emphasizing its role in mitochondrial homeostasis. Altogether, the results establish a role of Hsp22 in proteostasis and highlight the role of the UPRMT in preventing the aging process Source of funding: Supported by the Canadian Institutes for Health Research S114 LIFE ON PAUSE: EPIGENETIC MECHANISMS UNDERLIE METABOLIC STASIS IN COLD-ADAPTED ANIMALS K. Storey. Carleton University, Institute of Biochemistry and Department of Biology, Ottawa, Ontario, Canada For many animals, survival of severe environmental stress (e.g., cold, drought, oxygen limitation, food deprivation) is aided by entry into a hypometabolic state. Strong metabolic rate depression, often to only 1-20% of normal resting rate, is the core survival principle of adaptive strategies including hibernation, freeze tolerance, estivation, and anaerobiosis. Global biochemical controls are used to suppress and reprioritize energy use. Recently, we have been exploring the idea that mechanisms previously associated with epigenetic regulation contribute to global suppression of gene expression during all forms of hypometabolism. Coordinated changes in DNA and histone posttranslational modification (e.g., acetylation, methylation, phosphorylation), changes in the activities of histone deacetylases and DNA methyltransferases, and differential expression of microRNA species all appear to underlie the reorganization of cells as they
430
Abstracts / Cryobiology 73 (2016) 399e443
enter hypometabolic states. These modifications are readily reversible and should now be considered as crucial targets to explore and apply to induce stasis in transplantable organs. Epigenetic controls also work hand in hand with the formation of ribonuclear protein bodies in the nucleus and cytoplasm that sequester mRNA transcripts into storage until animals arouse again to normal metabolic function. This research, suggesting a new layer of regulatory control that can contribute to coordinating metabolic rate depression will provide new tools in the arsenal of cryomedical science. For more information visit www.kenstoreylab.com. Source of funding: Supported by NSERC Canada. S115 SURVIVING WINTER: NFATS REGULATE CRYOPROTECTION IN FREEZETOLERANT RANA SYLVATIC R. Al-attar*, K. Storey. Carleton University, Ottawa, Ontario, Canada * Corresponding author.
Cold temperatures can be detrimental to organisms’ health. The freezetolerant Rana sylvatica can survive prolonged periods of whole body freezing (60-75%), which halts a majority of biochemical and physiological processes including breathing before resuming function upon thawing. Freezing prevents oxygen delivery to organs and draws water from the cells to the extracellular matrix for ice crystal formation resulting in anoxia and dehydration. As a result, physiological and biochemical adaptations are crucial for survival during freezing. Some of these adaptations include producing large quantities of glucose (~300 mM), up-regulating stress responsive genes, and down-regulating metabolic rates to 10-20% of normal conditions. Metabolic regulations occur at the transcriptional, translational, and post-translational levels. This study aimed to understand the transcriptional regulation of the nuclear factor of activated T cell 1-4 (NFATs) family of transcription factors in liver and skeletal muscle during the freeze/thaw cycle in Rana sylvatica. Using western blotting and DNA binding assays, we showed that freezing causes differential regulation of NFAT proteins and NFAT DNA binding during 24 h freezing and 8 h thawing in both tissues. The increase in NFATc3 total proteins, nuclear localization and DNA binding activity in the liver may be responsible for inducing the activation of signal transducer and activator of transcription 3 (p-STAT3-S727). Activated STAT3 caused the transcriptional inhibition of the glycogen synthase kinase 3b gene, previously reported to be active in the liver during freezing. In comparison, NFATc3 in skeletal muscle showed no change in protein levels or DNA binding. Interestingly, osteopontin, a downstream target of NFATc3, showed a significant down-regulation in protein expression during 24 h freezing in the skeletal muscle, which may explain the absence of p-STAT3-S727 proteins in this tissue. Together, the data suggests that Rana sylvatica liver, unlike skeletal muscle, actively regulates targets that are involved in glucose metabolism during freezing. Source of funding: This study is funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) discovery grant (Funding #:6793) awarded to Dr. Kenneth B. Storey. S116 HIBR-MIRS: COLD-SENSITIVE NOVEL MICRORNA IN THE HIBERNATING 13-LINED GROUND SQUIRREL B. Luu 1, *, K. Biggar 1, C. Wu 2, K. Storey 1. 1 Carleton University, Ottawa, Ontario, Canada; 2 Gainesville, Florida, United States * Corresponding author.
As thirteen-lined ground squirrels (Ictidomys tridecemlineatus) enter hibernation, they characteristically switch their primary fuel source from carbohydrates to lipids, depress their basal metabolic rate by 96-e98% to conserve vital energy stores, and significantly drop their core body temperature to 0-e5 C. In recent years, research has demonstrated the importance of microRNA (small noncoding RNA molecules) in coldtolerant animal models, including their dynamic regulation throughout mammalian hibernation. MicroRNA are highly conserved among vertebrates, and function by post-transcriptionally regulating mRNA translation to adapt cells for survival. This study uses small RNA sequencing and
custom bioinformatics pipelines to identify novel species-specific microRNA, and to predict their function. A group of 17 novel microRNA were identified in the ground squirrel (Ictidomys tridecemlineatus), and their relative expression was quantified using real-time PCR in the liver, skeletal muscle, and heart tissues over four experimental conditions that represent the torpor-arousal cycle (euthermic in cold room, early torpor, late torpor, and interbout arousal). These novel microRNAs lacked homology to any other known microRNA in miRBase (the microRNA annotation database). The predicted mRNA targets of these novel microRNA were found to be enriched in biological processes that are known to be regulated during hibernation, such as lipid metabolism, ion-transport ATPases, and various cellular signaling cascades. This study is the first of its kind to identify, quantify, and predict the likely function of species-specific microRNA in a hibernating animal. Source of funding: This work was supported by a Discovery grant from the Natural Sciences and Engineering Research Council (NSERC) of Canada (grant# 6793). K.B.S. holds the Canada Research Chair in Molecular Physiology, B.E.L. holds an NSERC Canada Graduate Scholarship, K.K.B. holds an NSERC Banting postdoctoral fellowship, and CWW holds an NSERC postdoctoral fellowship. S117 BENEFICIAL EFFECTS OF GLUTATHIONE SUPPLEMENT DURING VITRIFICATION OF MOUSE OOCYTES AT THE GERMINAL VESICLE STAGE ON THEIR PREIMPLANTATION DEVELOPMENT FOLLOWING MATURATION AND FERTILIZATION IN VITRO A. Moawad 1, 2, 3, *, S. Tan 1, T. Taketo 1. 1 McGill University, Department of Obstetrics and Gynecology, Canada; 2 Department of Surgery, OriginELLE Fertility Clinic, Montreal Canada; 3 Cairo University, Department of Theriogenology, Giza, Egypt * Corresponding author. McGill University, Department of Obstetrics and Gynecology, Canada.
Cryopreservation of oocytes at the germinal vesicle (GV) stage is an attractive option in ART when immediate IVF or controlled-hormone stimulation is unavailable for a patient; however, the need of IVM diminishes blastocyst development from GV-oocytes and its applications. We previously reported that supplementation of the media with L-carnitine (LC) for vitrification, thawing, and IVM of GV-oocytes improves preimplantation development following IVF in (B6.DBA)F1 and B6 mice. LC has dual biological activities, lipid metabolism and antioxidant, and which activity contributes to the beneficial effects of LC during GV-oocyte cryopreservation remains unknown. The aim of this study was to investigate the effects of glutathione (GSH; a potent antioxidant) supplementation during vitrification and thawing of mouse GV-oocytes on preimplantation development following maturation and fertilization in vitro. Combined effects of GSH and LC during vitrification and IVM were also tested. Our results showed that GSH supplementation in the IVM medium exhibited no inhibitory effects on the cleavage or blastocyst development rate in both mouse strains except that GSH at 1 mM increased the blastocyst development rate in B6 mice. By contrast, GSH supplementation at 1 mM in vitrification and warming media significantly increased blastocyst development rates in both mouse strains. When GSH at 0.5 or 1.0 mM was combined with LC at 1.86 or 3.72 mM in vitrification and warming media, little improvement was found over GSH supplementation alone. However, LC supplementation at 1.86 mM during IVM following vitrification and warming of GV-ooctyes in the media supplemented with both GSH and LC at 0.5 and 3.72 mM, respectively, increased the blastocyst development rate in the (B6.DBA)F1 strain. We conclude that GSH supplementation alone during vitrification and warming of GV-oocytes improves the developmental competence of oocytes, suggesting that the antioxidant activity is important for reducing the damage inflicted by freezing. S118 COLD-SENSITIVE ION CHANNELS ARE INVOLVED IN CHILLING INJURY OF PIG OOCYTES K. Edashige 1, *, Y. Iwahara 1, S. Nariai 1, Y. Nishiya 1, M. Kitayama 1, S. Niimi 1, S. Seki 2, C. Koshimoto 3, K. Matsukawa 1, M. Kasai 1. 1 Kochi