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Cold-sensitive ion channels are involved in chilling injury of pig oocytes
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
Abstracts / Cryobiology 73 (2016) 399e443
University, Kochi, Kochi Prefecture, Japan; 2 Akita University, Akita, Akita Prefecture, Japan; 3 University of Miyazaki, Miyazaki, Miyazaki Prefecture, Japan * Corresponding author.
Pig oocytes are sensitive to cold temperature lower than 15 C. Pig ovaries collected at the slaughterhouse need to be kept at over 18e-25 C during transport to the laboratory in order to maintain the developmental ability of the oocytes. However, the reason why pig oocytes are quite sensitive to cold temperature has not been elucidated. Transient receptor potential (TRP) channels are a group of ion channels mostly located on the plasma membrane in many types of animal cells. Many of these channels mediate a variety of sensations, including warmth and coldness. In this study, we examined whether cold-sensitive TRP channels were involved in the chilling injury of pig oocytes. Pig oocytes at the maturing stage were used. In one set of experiments, oocytes at the immature and mature stages were also used. First, we examined the expression of mRNA of TRPA1 (sensitive to 17 C) in oocytes by real time-PCR. Next, we examined whether a specific inhibitor for TRPA1 (AP-18) attenuated chilling-injury of maturing oocytes by cold-treatment (15 C for 15 min). Finally, we suppressed the expression of TRPA1 in maturing oocytes by injecting double-stranded RNA of TRPA1 into immature oocytes, and examined whether the suppression of TRPA1 expression attenuated chilling-injury of maturing oocytes by cold-treatment. TRPA1 mRNA was expressed in immature, maturing, and mature oocytes. A TRPA1-inhibitor increased the rates of survival and maturation of maturing oocytes after cold-treatment. Furthermore, the suppression of the expression of TRPA1 also increased the rates of maturing oocytes after cold-treatment. These results strongly suggest that the sensation of coldness by cold-sensitive TRP channels is the trigger for chilling injury of pig oocytes. Our results provide important information for understanding chilling injury of pig oocytes. Source of funding: This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI). S119 VITRIFICATION OF MOUSE ZYGOTES; EFFECT OF RAPID WARMING S. Seki*, K. Basaki, Y. Komatsu, Y. Fukuda, M. Yano, T. Obata, Y. Matsuda, K. Nishijima. Akita University, Akita, Akita Prefecture, Japan * Corresponding author.
Previous findings with Peter Mazur on the vitrification of mouse oocytes by using cryotops demonstrated a high survival even when subjected to a moderate cooling rate and a vitrification solution containing only half the usual concentration of cryoprotectants, given that the warming rate is extremely high. Recently, genome-editing technology involving microinjection of CRISPR/Cas9 nucleases into one-cell zygotes is developing rapidly. In embryo banking, cryotubes are widely used. Although mouse embryos at the morula stage can be cryopreserved efficiently in cryotubes, one-cell zygotes cannot because of their sensitivity to cryoprotectant toxicity. Therefore, we vitrified mouse zygotes in cryotubes and examined the effects of the warming rate and the concentration of cryoprotectants on their survival. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20%, 30%, and 40% v/v respectively), ficoll, and sucrose. After exposure to the EFS solutions for 2 min at 23 C, 5 ul from each solution including the mouse zygotes was vitrified in a cryotube by direct immersion into liquid nitrogen. The cryotubes were then warmed moderately by being held in the air at 23 C for 1 min to avoid fracture damage. Subsequently, they were warmed by holding them at 23 C for 3 min (34 C/min), by adding 1 ml of a solution containing 0.5 M sucrose at 23 C (4,600 C/min), or by adding 1 ml sucrose solution at 37 C (6,600 C/min). With EFS40, the survival was low by cryoprotectant toxicity regardless of the warming rate. With EFS30 and EFS20, the survival was low if the warming rate was low, but the survival increased with higher warming rates. Lastly, with EFS20 and rapid warming, almost all of the zygotes developed to blastocysts in vitro. In conclusion, we have developed a vitrification method for mouse zygotes using cryotubes, which yields a high zygote survival after rapid warming.
431
S120 A CHEMICALLY DEFINED MEDIUM WITHOUT ANIMAL PRODUCTS FOR RABBIT EMBRYO VITRIFICATION M. Teixeira*, L. Commin, L. Gavin-plagne, P. Bruyere, A. Philibert, S. Buff, T. Joly. University of Lyon, Vetagro Sup, Marcy-l'Etoile, France * Corresponding author.
Embryo cryopreservation often requires the use of animal serum products, which represent a sanitary risk, and contain undefined material that can considerably vary between batches, altering its thermodynamic properties. CRYO3® is a chemically defined substitute without animal products, created for mononuclear cell cryopreservation. Our objective was to compare three rabbit embryo vitrification buffer media: IMV holding medium®: a commercial medium containing animal derived products (G1); D-PBS supplemented with 20% of CRYO3® (G2); and a CRYO3® medium (G3). All the media contained the same cryoprotectant composition. New Zealand does were superovulated and collected embryos (n ¼ 231) were randomly divided into three groups. After equilibration, embryos were exposed for 30 sec to a vitrification solution of G1/G2/G3 medium, containing 20% Me2SO and 20% EG, before being loaded to a Fibreplug (CVM kit, Cryologic) and vitrified by solid surface vitrification. Thawing was performed by immersing the end of the Fibreplug directly into a thawing solution (0.5 M sucrose, respectively), for 5 min, followed by three successive dilution baths. Embryos were cultured to the hatching stage in M199 medium (G1:52, G2:37, and G3:71 embryos), supplemented with 10% FCS (38.5 C, 5% CO2). The survival rate (non-damaged embryos after thawing per vitrified embryos), the blastocyst formation rate, and the hatching rate (per survived) were evaluated. The survival rate (G1:87%, G2:81% and G3:96%) was significantly superior in the CRYO3® group (p<0.05). No significant difference was observed regarding the blastocyst formation rate (respectively, 77%, 65% and 66%) or the hatching rate (35%, 19% and 24%). In conclusion, CRYO3® can be used as a chemically defined substitute for animal-based products in rabbit embryo vitrification solutions, reducing the solutions’ variability and the sanitary risk inherent to animal derived products. Source of funding: National Network of Biological Resources Centers CRBANIM (ANR11-INSB-0003) S121 OPTIMAL PROTOCOL OF CRYOPROTECTANT ADDING/REMOVAL FOR OVARIAN TISSUE: EFFECT OF MANNITOL I. Trutaieva*, V. Kiroshka, T. Bondarenko. Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, Ukraine * Corresponding author.
Currently, one of the tasks to improve the protocol efficiency for ovarian tissue cryopreservation is the reduction of ice crystal formation by increasing the cryoprotective agents (CPAs) concentration. The research aim was to assess the efficiency of the media containing osmotically active sugars (sucrose, mannitol) at a stepwise adding/removing of 3 M dimethyl sulphoxide (Me2SO) and propanediol (PROH) on preservation of ovarian tissue structure. It is shown that the presence of sugars in the adding media (200 mМ sucrose) and removing media (DMEM 250 mM sucrose or DMEM + 500 mM mannitol) prevented cell swelling and enabled the maintaining of tight cell-to-cell contacts in the tissue structure. The analysis of morphology showed that the level of injuries after addition/ removal was dependent on the CPA type and its removal mode. We demonstrated that Me2SO with more rapid penetration into the ovarian tissue less damaging to follicles. When using the CPA with lower penetration rate (PROH) there was shown different efficacies of sugars in dilution media. In the presence of sucrose there was observed an occurrence of damage, manifested in accumulation of fluid and disturbance of dense cell-to-cell contacts in the tissue structure. Our results indicated that mannitol had been more effective when we used 3 M PROH. Therefore, despite the fact that both sucrose and mannitol are the osmotically active components of the solutions used to addition/removal CPA, their effect is
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
Abstracts / Cryobiology 73 (2016) 399e443
University, Kochi, Kochi Prefecture, Japan; 2 Akita University, Akita, Akita Prefecture, Japan; 3 University of Miyazaki, Miyazaki, Miyazaki Prefecture, Japan * Corresponding author.
Pig oocytes are sensitive to cold temperature lower than 15 C. Pig ovaries collected at the slaughterhouse need to be kept at over 18e-25 C during transport to the laboratory in order to maintain the developmental ability of the oocytes. However, the reason why pig oocytes are quite sensitive to cold temperature has not been elucidated. Transient receptor potential (TRP) channels are a group of ion channels mostly located on the plasma membrane in many types of animal cells. Many of these channels mediate a variety of sensations, including warmth and coldness. In this study, we examined whether cold-sensitive TRP channels were involved in the chilling injury of pig oocytes. Pig oocytes at the maturing stage were used. In one set of experiments, oocytes at the immature and mature stages were also used. First, we examined the expression of mRNA of TRPA1 (sensitive to 17 C) in oocytes by real time-PCR. Next, we examined whether a specific inhibitor for TRPA1 (AP-18) attenuated chilling-injury of maturing oocytes by cold-treatment (15 C for 15 min). Finally, we suppressed the expression of TRPA1 in maturing oocytes by injecting double-stranded RNA of TRPA1 into immature oocytes, and examined whether the suppression of TRPA1 expression attenuated chilling-injury of maturing oocytes by cold-treatment. TRPA1 mRNA was expressed in immature, maturing, and mature oocytes. A TRPA1-inhibitor increased the rates of survival and maturation of maturing oocytes after cold-treatment. Furthermore, the suppression of the expression of TRPA1 also increased the rates of maturing oocytes after cold-treatment. These results strongly suggest that the sensation of coldness by cold-sensitive TRP channels is the trigger for chilling injury of pig oocytes. Our results provide important information for understanding chilling injury of pig oocytes. Source of funding: This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (KAKENHI). S119 VITRIFICATION OF MOUSE ZYGOTES; EFFECT OF RAPID WARMING S. Seki*, K. Basaki, Y. Komatsu, Y. Fukuda, M. Yano, T. Obata, Y. Matsuda, K. Nishijima. Akita University, Akita, Akita Prefecture, Japan * Corresponding author.
Previous findings with Peter Mazur on the vitrification of mouse oocytes by using cryotops demonstrated a high survival even when subjected to a moderate cooling rate and a vitrification solution containing only half the usual concentration of cryoprotectants, given that the warming rate is extremely high. Recently, genome-editing technology involving microinjection of CRISPR/Cas9 nucleases into one-cell zygotes is developing rapidly. In embryo banking, cryotubes are widely used. Although mouse embryos at the morula stage can be cryopreserved efficiently in cryotubes, one-cell zygotes cannot because of their sensitivity to cryoprotectant toxicity. Therefore, we vitrified mouse zygotes in cryotubes and examined the effects of the warming rate and the concentration of cryoprotectants on their survival. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20%, 30%, and 40% v/v respectively), ficoll, and sucrose. After exposure to the EFS solutions for 2 min at 23 C, 5 ul from each solution including the mouse zygotes was vitrified in a cryotube by direct immersion into liquid nitrogen. The cryotubes were then warmed moderately by being held in the air at 23 C for 1 min to avoid fracture damage. Subsequently, they were warmed by holding them at 23 C for 3 min (34 C/min), by adding 1 ml of a solution containing 0.5 M sucrose at 23 C (4,600 C/min), or by adding 1 ml sucrose solution at 37 C (6,600 C/min). With EFS40, the survival was low by cryoprotectant toxicity regardless of the warming rate. With EFS30 and EFS20, the survival was low if the warming rate was low, but the survival increased with higher warming rates. Lastly, with EFS20 and rapid warming, almost all of the zygotes developed to blastocysts in vitro. In conclusion, we have developed a vitrification method for mouse zygotes using cryotubes, which yields a high zygote survival after rapid warming.
431
S120 A CHEMICALLY DEFINED MEDIUM WITHOUT ANIMAL PRODUCTS FOR RABBIT EMBRYO VITRIFICATION M. Teixeira*, L. Commin, L. Gavin-plagne, P. Bruyere, A. Philibert, S. Buff, T. Joly. University of Lyon, Vetagro Sup, Marcy-l'Etoile, France * Corresponding author.
Embryo cryopreservation often requires the use of animal serum products, which represent a sanitary risk, and contain undefined material that can considerably vary between batches, altering its thermodynamic properties. CRYO3® is a chemically defined substitute without animal products, created for mononuclear cell cryopreservation. Our objective was to compare three rabbit embryo vitrification buffer media: IMV holding medium®: a commercial medium containing animal derived products (G1); D-PBS supplemented with 20% of CRYO3® (G2); and a CRYO3® medium (G3). All the media contained the same cryoprotectant composition. New Zealand does were superovulated and collected embryos (n ¼ 231) were randomly divided into three groups. After equilibration, embryos were exposed for 30 sec to a vitrification solution of G1/G2/G3 medium, containing 20% Me2SO and 20% EG, before being loaded to a Fibreplug (CVM kit, Cryologic) and vitrified by solid surface vitrification. Thawing was performed by immersing the end of the Fibreplug directly into a thawing solution (0.5 M sucrose, respectively), for 5 min, followed by three successive dilution baths. Embryos were cultured to the hatching stage in M199 medium (G1:52, G2:37, and G3:71 embryos), supplemented with 10% FCS (38.5 C, 5% CO2). The survival rate (non-damaged embryos after thawing per vitrified embryos), the blastocyst formation rate, and the hatching rate (per survived) were evaluated. The survival rate (G1:87%, G2:81% and G3:96%) was significantly superior in the CRYO3® group (p<0.05). No significant difference was observed regarding the blastocyst formation rate (respectively, 77%, 65% and 66%) or the hatching rate (35%, 19% and 24%). In conclusion, CRYO3® can be used as a chemically defined substitute for animal-based products in rabbit embryo vitrification solutions, reducing the solutions’ variability and the sanitary risk inherent to animal derived products. Source of funding: National Network of Biological Resources Centers CRBANIM (ANR11-INSB-0003) S121 OPTIMAL PROTOCOL OF CRYOPROTECTANT ADDING/REMOVAL FOR OVARIAN TISSUE: EFFECT OF MANNITOL I. Trutaieva*, V. Kiroshka, T. Bondarenko. Institute for Problems of Cryobiology & Cryomedicine of the National Academy of Sciences of Ukraine, Kharkov, Ukraine * Corresponding author.
Currently, one of the tasks to improve the protocol efficiency for ovarian tissue cryopreservation is the reduction of ice crystal formation by increasing the cryoprotective agents (CPAs) concentration. The research aim was to assess the efficiency of the media containing osmotically active sugars (sucrose, mannitol) at a stepwise adding/removing of 3 M dimethyl sulphoxide (Me2SO) and propanediol (PROH) on preservation of ovarian tissue structure. It is shown that the presence of sugars in the adding media (200 mМ sucrose) and removing media (DMEM 250 mM sucrose or DMEM + 500 mM mannitol) prevented cell swelling and enabled the maintaining of tight cell-to-cell contacts in the tissue structure. The analysis of morphology showed that the level of injuries after addition/ removal was dependent on the CPA type and its removal mode. We demonstrated that Me2SO with more rapid penetration into the ovarian tissue less damaging to follicles. When using the CPA with lower penetration rate (PROH) there was shown different efficacies of sugars in dilution media. In the presence of sucrose there was observed an occurrence of damage, manifested in accumulation of fluid and disturbance of dense cell-to-cell contacts in the tissue structure. Our results indicated that mannitol had been more effective when we used 3 M PROH. Therefore, despite the fact that both sucrose and mannitol are the osmotically active components of the solutions used to addition/removal CPA, their effect is