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To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20–40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 μL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.Rapid warming (18 467 °C/min) of vitrified one-cell rat embryos results in high rates of development to blastocysts (58.1%) and pups (50.0%), even with cryotubes.

While group-2 innate lymphoid cells (ILC2s) are highly proliferative in allergic inflammation, the removal of overactivated ILC2s in allergic diseases has not been investigated. We previously showed that chronic airway allergy induces “exhausted-like” dysfunctional ILC2s expressing T cell immunoreceptor with Ig and ITIM domains (TIGIT). However, the physiological relevance of these cells in chronic allergy remains elusive. To precisely identify and monitor TIGIT+ ILC2s, we generated TIGIT lineage tracer mice. Chronic allergy stably induced TIGIT+ ILC2s, which were highly activated, apoptotic, and were quickly removed from sites of chronic allergy. Transcripts from coding genes were globally suppressed in the cells, possibly due to reduced chromatin accessibility. Cell death in TIGIT+ ILC2s was enhanced by interactions with CD155 expressed on macrophages, whereas genetic ablation of Tigit or blockade by anti-TIGIT antagonistic antibodies promoted ILC2 survival, thereby deteriorating chronic allergic inflammation. Our work demonstrates that TIGIT shifts the fate of ILC2s toward activation-induced cell death, which could present a new therapeutic target for chronic allergies.

Thepermeability of cells is important for cryopreservation. Previously, we showed in mice that the permeability to water and cryoprotectants of oocytes and embryos at early cleavage stages (early embryos) is low because these molecules move across the plasma membrane predominantly by simple diffusion through the lipid bilayer, whereas permeability of morulae and blastocysts is high because of a water channel, aquaporin 3 (AQP3). In this study, we examined the pathways for the movement of water and cryoprotectants in bovine oocytes/embryos and the role of AQP3 in the movement by determining permeability, first in intact bovine oocytes/embryos, then in bovine morulae with suppressed AQP3 expression, and finally in mouse oocytes expressing bovine AQP3. Results suggest that water moves through bovine oocytes and early embryos slowly by simple diffusion, as is the case in mice, although channel processes are also involved in the movement. On the other hand, water appears to move through morulae and blastocysts predominantly by facilitated diffusion via channels, as in mice. Like water, cryoprotectants appear to move through bovine oocytes/early embryos mostly by simple diffusion, but channel processes could also be involved in the movement of glycerol and ethylene glycol, unlike that in mice. In bovine morulae, although glycerol and ethylene glycol would move predominantly by facilitated diffusion, mostly through AQP3, as in mice, dimethylsulfoxide appears to move predominantly by simple diffusion, unlike in mice. These results indicate that permeability-related properties of bovine oocytes/embryos are similar to those of mouse oocytes/embryos, but species-specific differences do exist.

Our studies the past 5 yr have concentrated on intracellular ice formation (IIF) in mature mouse oocytes at the metaphase stage of meiosis II. Here we report an analogous investigation of the temperature of intracellular ice nucleation in preimplantation embryo stages from one-cell to early morula suspended in 1 M ethylene glycol/PBS and cooled at 20°C/min to −70°C. Physical modeling indicates that oocytes and preimplantation embryos undergo very little osmotic shrinkage at that cooling rate. As a consequence, their interior becomes increasingly supercooled until the supercooling is abruptly terminated by IIF. Four categories of IIF were observed. The first two were 1) those undergoing IIF at temperatures well below the temperature of external ice formation (EIF; −7.2°C) vs. 2) those undergoing IIF within 1°C of the EIF temperature. The other two categories were those multicellular stages in which 3) all the blastomeres underwent IIF simultaneously vs. 4) those in which blastomeres underwent IIF sequentially. Embryos in categories 1 and 3 constituted the majority (80–90%), and for them, the mean IIF temperatures of one-cell, two-cell, four- to six-cell, and early eight-cell ranged from −37°C to −43°C, temperatures that indicate that IIF is a consequence of homogeneous nucleation. However, the IIF nucleation temperature of early morulae in categories 1 and 3 was markedly higher; namely, −23.1 ± 1.5°C. This marked rise in nucleation temperature coincides with the appearance of aquaporin 3 and gap junctions in early morulae (compacted eight-cell), and is presumably causally related.

For the cryopreservation of embryos, vitrification has various advantages, but it also has disadvantages because embryos are vitrified with a considerable supercooling (i.e., in nonequilibrium). Here, we tried to develop a novel method in which embryos are vitrified in near-equilibrium. The extent of equilibrium was assessed by examining whether vitrified embryos survive after being kept at −80°C. Two-cell embryos of ICR mice were vitrified with ethylene glycol (EG)-based solutions, either EFSa or EFSc solutions, which were mixtures of EG (30%–40%) and an FSa or FSc solution, respectively. The FSa and FSc solutions were PB1 medium containing 30% Ficoll plus 0.5 or 1.5 M sucrose, respectively. In vitro survival rate was high when embryos vitrified with 30%–40% EG (EFS30a, EFS40a, EFS30c, and EFS40c) were warmed rapidly. When embryos were vitrified and then kept at −80°C for 4 days, large proportions survived with EFS30c and EFS40c. When embryos were vitrified with EFS35c or EFS40c, the survival rate was high even for those kept at −80°C for 10 days. When embryos of ICR and C57BL/6J mice were vitrified with EFS35c or EFS40c and then kept at −80°C for 4 days, the survival rate was high even after recooling in liquid nitrogen; a high proportion (75%) of C57BL/6J embryos vitrified with EFS35c developed to term after transfer. In conclusion, we have developed a novel method by which embryos are vitrified in near-equilibrium. This will be a supreme method for cryopreservation, retaining the advantages of both current vitrification and equilibrium slow freezing.

Successful cryopreservation demands there be little or no intracellular ice. One procedure is classical slow equilibrium freezing, and it has been successful in many cases. However, for some important cell types, including some mammalian oocytes, it has not. For the latter, there are increasing attempts to cryopreserve them by vitrification. However, even if intracellular ice formation (IIF) is prevented during cooling, it can still occur during the warming of a vitrified sample. Here, we examine two aspects of this occurrence in mouse oocytes. One took place in oocytes that were partly dehydrated by an initial hold for 12 min at −25°C. They were then cooled rapidly to −70°C and warmed slowly, or they were warmed rapidly to intermediate temperatures and held. These oocytes underwent no IIF during cooling but blackened from IIF during warming. The blackening rate increased about 5-fold for each five-degree rise in temperature. Upon thawing, they were dead. The second aspect involved oocytes that had been vitrified by cooling to −196°C while suspended in a concentrated solution of cryoprotectants and warmed at rates ranging from 140°C/min to 3300°C/min. Survivals after warming at 140°C/min and 250°C/min were low (<30%). Survivals after warming at ≥2200°C/min were high (80%). When warmed slowly, they were killed, apparently by the recrystallization of previously formed small internal ice crystals. The similarities and differences in the consequences of the two types of freezing are discussed.

The permeability to water and cryoprotectants of the plasma membrane is crucial to the successful cryopreservation of embryos. Previously, we have shown in mouse morulae that water and glycerol move across the plasma membrane by facilitated diffusion, and we have suggested that aquaporin 3 plays an important role in their movement. In the present study, we clarify the contribution of aquaporin 3 to the movement of water and various cryoprotectants in mouse morulae by measuring the Arrhenius activation energies for permeability to cryoprotectants and water, through artificial expression of aquaporin 3 using Aqp3cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3at the one-cell zygote stage. The results show that aquaporin 3 plays an important role in the facilitated diffusion of water, glycerol, and ethylene glycol, but not of acetamide and dimethylsulfoxide. On the other hand, in a propylene glycol solution, aquaporin 3 in morulae transported neither propylene glycol nor water by facilitated diffusion, probably because of strong water-solute interactions. These results provide important information for understanding the permeability of the plasma membrane of the mouse embryo.

Destabilization of Atg7mRNA by the CCR4-NOT complex prevents p53-dependent cell death in the heart.

In many fish, including zebrafish, oocytes at the germinal vesicle stage (stage III) can be matured to the metaphase II stage (stage V) with the aid of maturation-inducing steroid in vitro. However, in mammals, it has been shown that nuclear maturation of oocytes dose not guarantee their ability to be fertilized and to develop to term, and that the maturation of the cytoplasm is essential for the ability. In fish, few reports are available on the cytoplasmic maturation of oocytes. In zebrafish oocytes, it has been reported that 60% or 75% Leibovitz L-15 medium containing 17alpha,20- beta-dihydroxy-4-pregnen-3-one (DHP) is effective for the nuclear maturation. However, when we matured zebrafish oocytes under the condition, most oocytes were not fertilized and did not hatch. Thus, the in vitro maturation method would not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable method for in vitro maturation of zebrafish oocytes by examining their ability to be fertilized and to develop to term. Zebrafish oocytes at the stage III were cultured in 50–100% Leibovitz L-15 medium (0.16–0.32 Osm/kg) supplemented with DHP; the highest rates of cleavage (24%, 18/76) and hatching (12%, 9/76) were obtained from oocytes matured in 90% Leibovitz L-15 medium (0.29 Osm/kg), but the rates of cleavage and hatching were low. Moreover, the fertilizable period of the oocytes was limited to only 10 min. In Atlantic croaker, turbot, salmonids, and common carp, it has been reported that the pH of fish ovarian fluids is not neutral but alkaline (pH 8.1–9.0). Therefore, we tried to determine the suitable pH of 90% Leibovitz L-15 medium for in vitro maturation. The rates of cleavage (45%, 45/100) and hatching (33%, 33/100) obtained in oocytes matured in the medium at pH 9.0 were significantly higher than those obtained in the medium at pH 7.5, 8.5, or 9.5 (cleavage rate, 16–29%; hatching rate, 8–21%). In addition, the fertilizable period of oocytes was extended to 80 min. Alkaline condition (pH 9.0) would facilitate normal maturation of zebrafish oocytes. However, the rates of cleavage (27–46%) and hatching (24–39%) were not high enough. To improve the fertilization rate of in vitro matured oocytes, we added BSA to the maturation medium, because it has been reported that BSA maintains the fertilizing ability of zebrafish oocytes when it is added to the medium for in vitro fertilization. The rates of cleavage (57– 70%) and hatching (48–63%) of oocytes matured in 90% Leibovitz L-15 medium at pH 9.0 containing 5 mg/ml BSA increased significantly. The addition of BSA to a maturation medium would be effective for acquiring the fertilizing ability of oocytes. Moreover, oocytes matured in the medium with BSA looked yellow, which was a typical appearance of spawned oocytes with good quality, whereas oocytes matured in a medium without BSA looked translucent. Therefore, BSA would also be effective for the maturation of zebrafish oocytes. In this study, we developed a reliable in vitro maturation system for zebrafish oocytes. This method will be a powerful tool for understanding the mechanism of maturation in zebrafish. (platform)

The permeability to water and cryoprotectants of the plasma membrane affects the suitable condition for cryopreservation of the cell. Previously, we showed that water, glycerol, ethylene glycol, DMSO, and acetamide move through mouse morulae by facilitated diffusion. We also showed that aquaporin 3, an aquaglyceroporin, plays an important role in the facilitated diffusion of water, glycerol and ethylene glycol, but that other channels are involved in the facilitated diffusion of DMSO and acetamide. In this study, we try to clarify whether aquaporin 9, another aquaglyceroporin, is involved in the movement of DMSO and acetamide first by suppressing the expression of aquaporin 9 in morulae by injecting double stranded RNA of mouse aquaporin 9 at the 1-cell zygote stage, and secondly by artificial expression of aquaporin 9 in oocytes. The suppression of the expression of aquaporin 9 in morulae significantly decreased the permeability to DMSO and acetamide, but not the permeability to water, glycerol and ethylene glycol, suggesting that aquaporin 9 plays an important role in the facilitated diffusion of DMSO and acetamide in morulae. The exogenous expression of aquaporin 9 in oocytes significantly increased the permeability to DMSO and acetamide. However, the exogenous expression also significantly increased the permeability to glycerol and ethylene glycol. It has been shown that cystic fibrosis transmembrane conductance regulator (CFTR) is a regulator of aquaporin 9 and can modulate the permeability-property of aquaporin 9. Therefore, we examined the expression of CFTR in oocytes and morulae. CFTR was markedly expressed in oocytes but not in morulae. Compared to the permeability of oocytes injected with aquaporin 9 cRNA alone, the additional injection of CFTR si-RNA significantly decreased the permeability to glycerol and ethylene glycol but not that to DMSO and acetamide. These results suggest that aquaporin 9 plays an important role in the movement of DMSO and acetamide in mouse morulae, and that aquaporin 9 also transports ethylene glycol and glycerol but only when CFTR is present. These results will provide important information for understanding the movement of water and cryoprotectants in mouse embryos. This research was supported by Grants-in-Aid for Scientific Research from JSPS (KAKENHI).