Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testes were vitrified with the solution. The post-vitrification survival (72.8 ± 3.5 %) was markedly improved compared with that achieved using the sucrose-containing solution (44.7 ± 4.2 %). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

Cryopreservation of rat embryos at all developmental stages by small-volume vitrification procedure and rapid warming in cryotubes.

Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 °C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30-100 µL at a cooling rate of 5830-7160 °C/min and warmed at 35,480-49,400 °C/min by adding 1 mL of 0.3 M sucrose solution at 50 °C, 17.3-28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 µl at 7950 °C/min and warmed at 68,850 °C/min, 58.8 ± 10.6% developed into blastocysts and 50.0 ± 7.4% developed to term, comparable to that of non-treated embryos (57.0 ± 5.4% and 51.4 ± 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.

TIGIT mediates activation-induced cell death of ILC2s during chronic airway allergy.

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.

Streamlined and quantitative detection of chimerism using digital PCR.

Animal chimeras are widely used for biomedical discoveries, from developmental biology to cancer research. However, the accurate quantitation of mixed cell types in chimeric and mosaic tissues is complicated by sample preparation bias, transgenic silencing, phenotypic similarity, and low-throughput analytical pipelines. Here, we have developed and characterized a droplet digital PCR single-nucleotide discrimination assay to detect chimerism among common albino and non-albino mouse strains. In addition, we validated that this assay is compatible with crude lysate from all solid organs, drastically streamlining sample preparation. This chimerism detection assay has many additional advantages over existing methods including its robust nature, minimal technical bias, and ability to report the total number of cells in a prepared sample. Moreover, the concepts discussed here are readily adapted to other genomic loci to accurately measure mixed cell populations in any tissue.

Small-volume vitrification and rapid warming yield high survivals of one-cell rat embryos in cryotubes†.

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.

Characteristic features of newly established specific pathogen-free albino large rabbit (JW-AKT): Comparison with Japanese White and New Zealand White rabbits.

The present study was conducted to reveal characteristic features of albino large rabbit (JW-AKT) which we formerly established a specific pathogen-free (SPF) colony. Body weights of JW-AKT rabbit at 52 weeks old was 5.7 ± 0.4 kg in males and 6.4 ± 0.4 kg in females. Weight of body, heart, lung and kidney in JW-AKT rabbit was significantly higher than in Japanese white and New Zealand white rabbits in both sexes. Though the body weight (BW) was rather lower in males, body length and brain weights tended to be higher in males than in females. Since body fat was significantly higher in females, what affects difference in BW is body fat, rather than the physical constitution of female JW-AKT rabbit. No critical sex difference was found in hematological parameters in JW-AKT rabbit. The results indicated that JW-AKT were about 1.5 times larger than the general laboratory rabbits with common properties in hematology. Thus, JW-AKT rabbit could be used as a novel SPF experimental animal model with some advantages in surgical experiments or collection of large amount of biological specimen.

Vitrification of one-cell mouse embryos in cryotubes.

Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-µl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.

The CCR4-NOT deadenylase complex controls Atg7-dependent cell death and heart function.

Shortening and removal of the polyadenylate [poly(A)] tail of mRNA, a process called deadenylation, is a key step in mRNA decay that is mediated through the CCR4-NOT (carbon catabolite repression 4-negative on TATA-less) complex. In our investigation of the regulation of mRNA deadenylation in the heart, we found that this complex was required to prevent cell death. Conditional deletion of the CCR4-NOT complex components Cnot1 or Cnot3 resulted in the formation of autophagic vacuoles and cardiomyocyte death, leading to lethal heart failure accompanied by long QT intervals. Cnot3 bound to and shortened the poly(A) tail of the mRNA encoding the key autophagy regulator Atg7. In Cnot3-depleted hearts, Atg7 expression was posttranscriptionally increased. Genetic ablation of Atg7, but not Atg5, increased survival and partially restored cardiac function of Cnot1 or Cnot3 knockout mice. We further showed that in Cnot3-depleted hearts, Atg7 interacted with p53 and modulated p53 activity to induce the expression of genes encoding cell death-promoting factors in cardiomyocytes, indicating that defects in deadenylation in the heart aberrantly activated Atg7 and p53 to promote cell death. Thus, mRNA deadenylation mediated by the CCR4-NOT complex is crucial to prevent Atg7-induced cell death and heart failure, suggesting a role for mRNA deadenylation in targeting autophagy genes to maintain normal cardiac homeostasis.

Production of the medaka derived from vitrified whole testes by germ cell transplantation.

The medaka (Oryzias latipes) is a teleost model distinguished from other model organisms by the presence of inbred strains, wild stocks, and related species. Cryopreservation guarantees preservation of these unique biological resources. However, because of their large size, cryopreservation techniques for their eggs and embryos have not been established. In the present study, we established a methodology to produce functional gametes from cryopreserved testicular cells (TCs). Whole testes taken from medaka were cryopreserved by vitrification. After thawing, the cells dissociated from cryopreserved testicular tissues were intraperitoneally transplanted into sterile triploid hatchlings. Some cells, presumably spermatogonial stem cells, migrated into the genital ridges of recipients and resulted in the production of eggs or sperm, based on sex of the recipient. Mating of recipients resulted in successful production of cryopreserved TC-derived offspring. We successfully produced individuals from the Kaga inbred line, an endangered wild population in Tokyo, and a sub-fertile mutant (wnt4b-/-) from cryopreserved their TCs. This methodology facilitates semi-permanent preservation of various medaka strains.

Intracellular ice formation in mouse zygotes and early morulae vs. cooling rate and temperature-experimental vs. theory.

In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to -70 °C at 0.5-20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ''flashing''. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of -7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as "high temperature'' flashers. In zygotes, there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below -7.7 °C; these we call ''low temperature'' flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes. Also, the mean flashing temperature is much higher with morulae (-20.9 °C) than with zygotes (-40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good.

Production of viable trout offspring derived from frozen whole fish.

Long-term preservation of fish fertility is essential for the conservation of endangered fishes. However, cryopreservation techniques for fish oocytes and embryos have not yet been developed. In the present study, functional eggs and sperm were derived from whole rainbow trout that had been frozen in a freezer and stored without the aid of exogenous cryoprotectants. Type A spermatogonia retrieved from frozen-thawed whole trout remained viable after freezing duration up to 1,113 days. Long-term-frozen trout spermatogonia that were intraperitoneally transplanted into triploid salmon hatchlings migrated toward the recipient gonads, where they were incorporated, and proliferated rapidly. Although all triploid recipients that did not undergo transplantation were functionally sterile, 2 of 12 female recipients and 4 of 13 male recipients reached sexual maturity. Eggs and sperm obtained from the salmon recipients were capable of producing donor-derived trout offspring. This methodology is thus a convenient emergency tool for the preservation of endangered fishes.

Extreme rapid warming yields high functional survivals of vitrified 8-cell mouse embryos even when suspended in a half-strength vitrification solution and cooled at moderate rates to -196°C.

To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to do so is to subject them to procedures that convert cell water into a non-crystalline glass. Current belief is that to achieve this vitrification, cells must be suspended in very high concentrations of glass-inducing solutes (i.e., ≥6 molal) and cooled at very high rates (i.e., ≫1000°C/min). We report here that both these beliefs are incorrect with respect to the vitrification of 8-cell mouse embryos. In this study, precompaction 8-cell embryos were vitrified in several dilutions of EAFS10/10 using various cooling rates and warming rates. Survival was based on morphology, osmotic functionality, and on the ability to develop to expanded blastocysts. With a warming rate of 117,500°C/min, the percentages of embryos vitrified in 1×, 0.75×, and 0.5× EAFS that developed to blastocysts were 93%, 92%, and 83%, respectively. And the percentages of morphological survivors that developed to expanded blastocysts were 100%, 92%, and 97%, respectively. Even when the solute concentration of the EAFS was reduced to 33% of normal, we obtained 40% functional survival of these 8-cell embryos.

A trial to cryopreserve immature medaka (Oryzias latipes) oocytes after enhancing their permeability by exogenous expression of aquaporin 3.

Fish oocytes have not been cryopreserved successfully, probably because it is difficult to prevent intracellular ice from forming. Previously, we have shown in medaka that immature oocytes are more suitable for cryopreservation than mature oocytes or embryos, in terms of permeability. We have also shown in immature medaka oocytes that the exogenous expression of aquaporin 3 (AQP3), a water/cryoprotectant channel, promotes the movement of water and cryoprotectants through the plasma membrane. In the present study, we attempted to cryopreserve immature medaka oocytes expressing AQP3. We first examined effects of hypertonic stress and the chemical toxicity of cryoprotectants on the survival of the AQP3-expressing oocytes. Exposure to hypertonic solutions containing sucrose decreased the survival of oocytes, but the expression of AQP3 did not affect sensitivity to hypertonic stress. Also, AQP3 expression did not markedly increase sensitivity to the toxicity of cryoprotectants. Of the four cryoprotectants tested, propylene glycol was the least toxic. Using a propylene glycol-based solution, therefore, we tried to cryopreserve immature oocytes by vitrification. During cooling with liquid nitrogen, all intact oocytes became opaque, but many AQP3-expressing oocytes remained transparent. This indicates that the expression of AQP3 is effective in preventing intracellular ice from forming during cooling. During warming, however, all the AQP3-expressing oocytes became opaque, indicating that intracellular ice formed. Therefore, the dehydration and permeation by propylene glycol were still insufficient. Further studies are necessary to realize the cryopreservation of fish oocytes.

Ultra-rapid warming yields high survival of mouse oocytes cooled to -196°c in dilutions of a standard vitrification solution.

Intracellular ice is generally lethal. One way to avoid it is to vitrify cells; that is, to convert cell water to a glass rather than to ice. The belief has been that this requires both the cooling rate and the concentration of glass-inducing solutes be very high. But high solute concentrations can themselves be damaging. However, the findings we report here on the vitrification of mouse oocytes are not in accord with the first belief that cooling needs to be extremely rapid. The important requirement is that the warming rate be extremely high. We subjected mouse oocytes in the vitrification solution EAFS 10/10 to vitrification procedures using a broad range of cooling and warming rates. Morphological survivals exceeded 80% when they were warmed at the highest rate (117,000°C/min) even when the prior cooling rate was as low as 880°C/min. Functional survival was >81% and 54% with the highest warming rate after cooling at 69,000 and 880°C/min, respectively. Our findings are also contrary to the second belief. We show that a high percentage of mouse oocytes survive vitrification in media that contain only half the usual concentration of solutes, provided they are warmed extremely rapidly; that is, >100,000°C/min. Again, the cooling rate is of less consequence.

Effect of the expression of aquaporins 1 and 3 in mouse oocytes and compacted eight-cell embryos on the nucleation temperature for intracellular ice formation.

The occurrence of intracellular ice formation (IIF) is the most important factor determining whether cells survive a cryopreservation procedure. What is not clear is the mechanism or route by which an external ice crystal can traverse the plasma membrane and cause the heterogeneous nucleation of the supercooled solution within the cell. We have hypothesized that one route is through preexisting pores in aquaporin (AQP) proteins that span the plasma membranes of many cell types. Since the plasma membrane of mature mouse oocytes expresses little AQP, we compared the ice nucleation temperature of native oocytes with that of oocytes induced to express AQP1 and AQP3. The oocytes were suspended in 1.0  M ethylene glycol in PBS for 15  min, cooled in a Linkam cryostage to -7.0  ° C, induced to freeze externally, and finally cooled at 20  ° C/min to -70  ° C. IIF that occurred during the 20  ° C/min cooling is manifested by abrupt black flashing. The mean IIF temperatures for native oocytes, for oocytes sham injected with water, for oocytes expressing AQP1, and for those expressing AQP3 were -34, -40, -35, and -25  ° C respectively. The fact that the ice nucleation temperature of oocytes expressing AQP3 was 10-15  ° C higher than the others is consistent with our hypothesis. AQP3 pores can supposedly be closed by low pH or by treatment with double-stranded Aqp3 RNA. However, when morulae were subjected to such treatments, the IIF temperature still remained high. A possible explanation is suggested.

Pathway for the movement of water and cryoprotectants in bovine oocytes and embryos.

The permeability 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.

Developmental ability of vitrified mouse oocytes expressing water channels.

Previously, we showed that the exogenous expression of aquaporin 3 (AQP3), an aquaglyceroporin, improved the tolerance of mouse oocytes to vitrification with a glycerol-based solution. In the present study, we examined conditions suitable for the expression of AQP3 and the ability of vitrified oocytes to develop in vitro and in vivo after fertilization. After only partial remove of cumulus cells, immature mouse oocytes (germinal vesicle stage) were injected with 5, 10 or 20 pg of AQP3 cRNA and cultured for 12 h for maturation. When matured oocytes were vitrified with a glycerol-based solution, 57-61% survived regardless of the amount of cRNA injected (5-20 pg). By contrast, no oocytes injected with water (control) survived. When the zona pellucida was removed from the vitrified oocytes and the oocytes were then fertilized in vitro and cultured, the proportions that were fertilized and developed into blastocysts were higher when the amount of cRNA injected was 5 pg than 10-20 pg. When 16 blastocysts were transferred to a pseudopregnant mouse, 5 developed to term, demonstrating that oocytes vitrified after injection of AQP3 cRNA retained the ability to develop to term. The water-permeability of cRNA-injected oocytes was higher than that of control oocytes from the maturing stage to the 1-cell zygote stage, whereas glycerol-permeability was higher only at metaphase II. This indicates that AQP3 was expressed for a relatively short period of time. These results suggest that the transient expression of water/cryoprotectant channels is effective for cryopreserving cells that have low membrane-permeability, such as mammalian oocytes.

Stability of mouse oocytes at -80 °C: the role of the recrystallization of intracellular ice.

The germplasm of mutant mice is stored as frozen oocytes/embryos in many facilities worldwide. Their transport to and from such facilities should be easy and inexpensive with dry ice at -79 °C. The purpose of our study was to determine the stability of mouse oocytes with time at that temperature. The metaphase II oocytes were cryopreserved with a vitrification solution (EAFS10/10) developed by M Kasai and colleagues. Two procedures were followed. In one, the samples were cooled at 187 °C/min to -196 °C, warmed to -80 °C, held at -80 °C for 1 h to 3 months, and warmed to 25 °C at one of three rates. With the highest warming rate (2950 °C/min), survival remained at 75% for the first month, but then slowly declined to 40% over the next 2 months. With the slowest warming (139 °C/min), survival was only ∼ 5% even at 0 time at -80 °C. In the second procedure, the samples were cooled at 294 °C/min to -80 °C (without cooling to -196 °C) and held for up to 3 months before warming at 2950 °C/min. Survival was ∼ 90% after 7 days and dropped slowly to 35% after 3 months. We believe that small non-lethal quantities of intracellular ice formed during the cooling and that the intracellular crystals increased to a damaging size by recrystallization during the 3 month's storage at -80 °C. From the practical point of view, this protocol yields sufficient stability to make it feasible to ship oocytes worldwide in dry ice.

Survival of mouse oocytes after being cooled in a vitrification solution to -196°C at 95° to 70,000°C/min and warmed at 610° to 118,000°C/min: A new paradigm for cryopreservation by vitrification.

There is great interest in achieving reproducibly high survivals of mammalian oocytes (especially human) after cryopreservation, but the results to date have not matched the interest. A prime cause of cell death is the formation of more than trace amounts of intracellular ice, and one strategy to avoid it is vitrification. In vitrification procedures, cells are loaded with high concentrations of glass-inducing solutes and cooled to -196°C at rates high enough to presumably induce the glassy state. In the last decade, several devices have been developed to achieve very high cooling rates. Nearly all in the field have assumed that the cooling rate is the critical factor. The purpose of our study was to test that assumption by examining the consequences of cooling mouse oocytes in a vitrification solution at four rates ranging from 95 to 69,250°C/min to -196°C and for each cooling rate, subjecting them to five warming rates back above 0°C at rates ranging from 610 to 118,000°C/min. In samples warmed at the highest rate (118,000°C/min), survivals were 70% to 85% regardless of the prior cooling rate. In samples warmed at the lowest rate (610°C/min), survivals were low regardless of the prior cooling rate, but decreased from 25% to 0% as the cooling rate was increased from 95 to 69,000°C/min. Intermediate cooling and warming rates gave intermediate survivals. The especially high sensitivity of survival to warming rate suggests that either the crystallization of intracellular glass during warming or the growth by recrystallization of small intracellular ice crystals formed during cooling are responsible for the lethality of slow warming.

Cryobiological properties of immature zebrafish oocytes assessed by their ability to be fertilized and develop into hatching embryos.

As a step to develop a cryopreservation method for zebrafish oocytes, we investigated the cryobiological properties of immature oocytes at stage III by examining their ability to mature and to develop into hatching embryos after fertilization. When oocytes were chilled at -5°C for 30min, the maturation rate decreased, but the rates of fertilization and hatching were not significantly different from those of controls. When oocytes were exposed to hypotonic solutions for 60min at 25°C, the rates of maturation, fertilization, and hatching decreased in a solution with 0.16Osm/kg or below. When oocytes were exposed to hypertonic solutions (containing sucrose) at 25°C for 30min, the maturation rate decreased in solution with 0.51Osm/kg, whereas the hatching rate decreased with lower osmolality (0.40Osm/kg). In an experiment on the toxicity of cryoprotectants (∼10%, at 25°C), it was found that glycerol and ethylene glycol were toxic both by the assessment of maturation and hatching. Propylene glycol, DMSO and methanol were less toxic by the assessment of maturation, but were found to be toxic by the assessment of hatching. Methanol was the least toxic, but it was less effective to make a solution vitrify than propylene glycol. Therefore, a portion of methanol was replaced with propylene glycol. The replacement increased the toxicity, but could be effective to reduce chilling injury at -5°C. These results clarified the sensitivity of immature oocytes to various cryobiological properties accurately, which will be useful for realizing cryopreservation of zebrafish oocytes.