Trp-tRNA synthetase bridges DNA-PKcs to PARP-1 to link IFN-γ and p53 signaling.

Interferon-γ (IFN-γ) engenders strong antiproliferative responses, in part through activation of p53. However, the long-known IFN-γ-dependent upregulation of human Trp-tRNA synthetase (TrpRS), a cytoplasmic enzyme that activates tryptophan to form Trp-AMP in the first step of protein synthesis, is unexplained. Here we report a nuclear complex of TrpRS with the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) and with poly(ADP-ribose) polymerase 1 (PARP-1), the major PARP in human cells. The IFN-γ-dependent poly(ADP-ribosyl)ation of DNA-PKcs (which activates its kinase function) and concomitant activation of the tumor suppressor p53 were specifically prevented by Trp-SA, an analog of Trp-AMP that disrupted the TrpRS-DNA-PKcs-PARP-1 complex. The connection of TrpRS to p53 signaling in vivo was confirmed in a vertebrate system. These and further results suggest an unexpected evolutionary expansion of the protein synthesis apparatus to a nuclear role that links major signaling pathways.

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.

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.

Production of functional eggs and sperm from in vitro-expanded type A spermatogonia in rainbow trout.

Combining cryopreservation of germline stem cells (GSCs) with their subsequent transplantation into recipient fish is a powerful tool for long-term preservation of genetic resources of endangered fishes. However, application of this technique has been limited because endangered species sometimes have small gonads and do not supply enough GSCs to be used for transplantation. This limitation could be overcome by expanding GSCs in vitro, though this has been difficult due to the complexity of reconstructing the gonadal microenvironment that surrounds GSCs. Here, we describe a novel method of in vitro expansion of rainbow trout GSCs using a feeder layer derived from Sertoli cells and a culture medium containing trout plasma. A transplantation assay demonstrated that the in vitro-expanded GSCs exhibited stem cell activity and potency to produce functional eggs, sperm, and eventually healthy offspring. In vitro expansion of GSCs can aid in rescuing fishes that are on the verge of extinction.

Development of a reliable in vitro maturation system for zebrafish oocytes.

In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, these in vitro maturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable in vitro maturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50-100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5-9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16-29%; hatching rate, 8-21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism of in vitro maturation in zebrafish oocytes and for the practical use of immature oocytes.

Formation of extracellular and intracellular ice during warming of vitrified mouse morulae and its effect on embryo survival.

In vitrified solutions, ice can form during warming if the concentration of the cryoprotectant is insufficient. For the cryopreservation of cells, ice is innocuous when it remains outside the cell, but intracellular ice (ICI) is lethal. We tried to estimate the conditions in which ICI forms in vitrified mouse morulae during warming. The solutions for the experiments (EFS10-EFS50) contained 10-50% ethylene glycol plus Ficoll plus sucrose. When vitrified EFS20, EFS30, and EFS40 were kept at -80 degrees C, they remained transparent after 3 min, but turned opaque after 60 min (EFS20, EFS30) or 24h (EFS40). Morulae were vitrified with EFS solutions after exposure for 30-120 s at 25 degrees C. They were warmed by various methods and survival was assessed in culture. After rapid warming (control), survival was high with EFS30 (79-93%) and EFS40 (96-99%). After slow warming, survival decreased with both EFS30 (48-62%) and EFS40 (44-64%). This must be from the formation of ICI. To examine the temperature at which ICI formed during slow warming, vitrified embryos were kept at various sub-zero temperatures during warming. Survival with EFS30 and EFS40 decreased on keeping samples for 3 min at -80 (25-75%), -60 (7-49%), -40 (0-41%), or -20 degrees C (26-60%). When samples were kept at -80 degrees C for 24h, the survival decreased to 0-14%. These results suggest that ICI forms at a wide range of temperatures including -80 and -20 degrees C, more likely between -60 and -40 degrees C, and the ice forms not only quickly but also slowly.

Sensitivity to chilling of medaka (Oryzias latipes) embryos at various developmental stages.

As an essential step toward cryopreservation of fish embryos, we examined the chilling sensitivity of medaka (Oryzias latipes) embryos at various developmental stages. Embryos at the 2-4 cell, 8-16 cell, morula, blastula, and early gastrula stages were suspended in Hanks solution. They were chilled to various temperatures (usually 0 degrees C), kept for various periods (usually 20 min), then cultured for up to 14 d to determine survival (assessed by the ability to hatch). Embryos at the 2-4 cell stage were the most sensitive to chilling to 0 degrees C, but sensitivity decreased as development proceeded. The survival rate of 2-4 cell embryos was affected after 2 min of chilling at 0 degrees C; although the rate decreased gradually as the duration of chilling increased, 38% of them still survived after 40 min of chilling. Embryos at the 2-4 cell stage were sensitive to chilling at 0 or -5 degrees C, but much less sensitive at 5 or 10 degrees C. The survival rate of 2-4 cell embryos subjected to repeated rapid cooling and warming was similar to that of those kept chilled. When early gastrula embryos were preserved at 0 or 5 degrees C, the hatching rate did not decrease after 12 and 24h of chilling, respectively, but then decreased gradually as storage was prolonged; however, 3-10% of the embryos hatched even after storage for 10 d. In conclusion, although later-stage medaka embryos would be suitable for cryopreservation (from the perspective of chilling sensitivity), chilling injury may not be serious in earlier stage embryos.

Embryonic senescence and laminopathies in a progeroid zebrafish model.

BACKGROUND: Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. PRINCIPAL FINDINGS: We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with relatively mild phenotypes only, but had shortened lifespan with obvious distortion of body shape. CONCLUSION: We generated new zebrafish models for a human premature aging disorder, and further demonstrated the utility for studying laminopathies. Premature aging could also be modeled in zebrafish embryos. This genetic model may thus provide a new platform for future drug screening as well as genetic analyses aimed at identifying modifier genes that influence not only progeria and laminopathies but also other age-associated human diseases common in vertebrates.

The mechanism by which mouse spermatozoa are injured during freezing.

To improve the cryopreservation protocol for mouse sperm, we attempted to estimate the type and extent of cryoinjury at various steps of the process. First, we demonstrated that mouse sperm are sensitive to chilling at -15 C and that the sensitivity is dependent on the length of exposure. To estimate cryoinjuries, sperm suspensions were ice-seeded at -5 or -15 C, frozen with liquid nitrogen (LN(2)) gas and then frozen in LN(2). In one experiment, sperm seeded at -5 C were cooled slowly to -15 C before deep freezing. At various steps of the cryopreservation process, the sperm were warmed and their viability was assessed based on motility and the integrities of the plasma membrane and acrosome. The motility of frozen-thawed sperm was higher on seeding at -5 C (28%) than at -15 C (9%). The motility did not decrease when the sample was transferred from LN(2) gas to LN(2). To estimate cryoinjury of sperm, we presumed the viability of frozen sperm to be decreased by chilling, hypertonic stress and formation of intracellular ice. When the sperm suspension was cooled and seeded at -5 C, the motility decreased by 25% due to hypertonic stress. When the sperm were cooled in LN(2) gas, the motility decreased by 17% with the formation of intracellular ice. When the sperm were cooled to -15 C, the motility decreased by 51% from chilling. After seeding, the motility decreased by 18% due to formation of intracellular ice and by 7% due to hypertonic stress. Considering the results, it would be preferable to seed samples at a higher temperature to prevent intracellular ice from forming and to cool seeded samples rapidly enough to minimize chilling injury and hypertonic stress, but not too rapidly to allow intracellular ice to form.

Exogenous expression of rat aquaporin-3 enhances permeability to water and cryoprotectants of immature oocytes in the zebrafish (Danio rerio).

Movement of water and cryoprotectants through the plasma membrane needs to be accelerated for successful cryopreservation of zebrafish oocytes/embryos, which are much larger than their mammalian counterparts. Aquaporin-3 is a water/solute channel that can transport not only water but also various cryoprotectants. In this study, we attempted to increase the permeability of immature zebrafish oocytes at stage III to water and cryoprotectants by exogenous expression of rat aquaporin-3. Immature zebrafish oocytes were injected with rat aquaporin-3 cRNA and cultured for 5-12 h. Permeability to water and cryoprotectants was then determined based on changes in the volumes of the oocytes in a hypertonic sucrose solution and various cryoprotectant solutions at 25 C. The permeability to water of the aquaporin-3 cRNA-injected oocytes was three times higher than that of intact and water-injected oocytes. The permeability of the aquaporin-3 cRNA-injected oocytes to ethylene glycol, glycerol, propylene glycol, and DMSO was also 2-4 times higher than that of intact oocytes. Thus, the permeability of immature zebrafish oocytes to water and cryoprotectants was enhanced by exogenous expression of aquaporin-3. Cryopreservation of teleost oocytes may be realized through a further increase in permeability.

The permeability to water and cryoprotectants of immature and mature oocytes in the zebrafish (Danio rerio).

To identify a stage feasible for the cryopreservation of zebrafish oocytes, we investigated the permeability to water and cryoprotectants of immature (stage III) and mature (stage V) oocytes. The permeability to water (microm/min/atm) of immature oocytes at 25 degrees C (0.37) was significantly higher than that of mature oocytes (0.10). The permeability (x10(-3)cm/min) of immature oocytes to ethylene glycol, propylene glycol, and Me(2)SO (1.49-3.03) at 25 degrees C was substantially higher than that of mature oocytes approximately 0. The permeability of immature oocytes to glycerol was also high (1.75), although the permeability could not be measured in mature oocytes. Immature oocytes would be more suitable than mature oocytes for conservation of the zebrafish.

The role of aquaporin 3 in the movement of water and cryoprotectants in mouse morulae.

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 Aqp3 cRNA in mouse oocytes, and by suppressing the expression of aquaporin 3 in morulae by injecting double-stranded RNA of Aqp3 at 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.

Expression of aquaporin-3 improves the permeability to water and cryoprotectants of immature oocytes in the medaka (Oryzias latipes).

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes and embryos. Several efforts have been made to facilitate the movement of water and cryoprotectants across the plasma membrane of fish oocytes/embryos because of their large size. Aquaporin-3 is a water/solute channel that can also transport various cryoprotectants. In this study, we tried to improve the permeability of immature medaka (Oryzias latipes) oocytes to water and cryoprotectants by artificially expressing aquaporin-3. The oocytes were injected with aquaporin-3 cRNA and cultured for 6-7 h. Then, hydraulic conductivity (L(P)) and cryoprotectant permeability (P(S)) were determined from volume changes in a hypertonic sucrose solution and various cryoprotectant solutions, respectively, at 25 degrees C. The L(P) value of the cRNA-injected oocytes was 0.22+/-0.04 microm/min/atm, nearly twice larger than that of intact or water-injected oocytes (0.14+/-0.02 and 0.14+/-0.03 microm/min/atm, respectively). P(S) values of intact oocytes for ethylene glycol, propylene glycol, and DMSO were 1.36+/-0.34, 1.97+/-0.20, and 1.17+/-0.52 x 10(-3) cm/min, respectively. The permeability to glycerol could not be calculated because oocytes remained shrunken in the glycerol solution. On the other hand, cRNA-injected oocytes had significantly higher P(S) values (glycerol, 2.20+/-1.29; ethylene glycol, 2.98+/-0.36; propylene glycol, 3.93+/-1.70; DMSO, 3.11+/-0.74 x 10(-3) cm/min) than intact oocytes. When cRNA-injected oocytes were cultured for 12-14 h, 51% matured to the metaphase II stage, and 43% of the matured oocytes were fertilized and hatched following in vitro fertilization and 14 days of culture. Thus, the permeability of medaka oocytes to water and cryoprotectants was improved by the artificial expression of aquaporin-3, and the oocytes retained the ability to develop to term.

Japanese flounder (Paralichthys olivaceus) embryos are difficult to cryopreserve by vitrification.

The first successful cryopreservation of fish embryos was reported in the Japanese flounder by vitrification [Chen and Tian, Theriogenology, 63, 1207-1219, 2005]. Since very high concentrations of cryoprotectants are needed for vitrification and fish embryos have a large volume, Japanese flounder embryos must have low sensitivity to cryoprotectant toxicity and high permeability to water and cryoprotectants. So, we investigated the sensitivity and the permeability of Japanese flounder embryos. In addition, we assessed the survival of flounder embryos after vitrification with solutions containing methanol and propylene glycol, following Chen and Tian's report. The embryos were relatively insensitive to the toxicity of individual cryoprotectants at lower concentrations, especially methanol and propylene glycol as their report. Although their permeability to water and cryoprotectants could not be measured from volume changes in cryoprotectant solutions, the embryos appeared to be permeable to methanol but less permeable to DMSO, ethylene glycol, and propylene glycol. Although vitrification solutions containing methanol and propylene glycol, which were used in Chen and Tian's report, were toxic to embryos, a small proportion of embryos did survived. However, when vitrified with the vitrification solutions, no embryos survived after warming. The embryos became opaque during cooling with liquid nitrogen, indicating the formation of intracellular ice during cooling. When embryos had been kept in vitrification solutions for 60 min after being treated with the vitrification solution, some remained transparent during cooling, but became opaque during warming. This suggests that dehydration and/or permeation by cryoprotectants were insufficient for vitrification of the embryos even after they had been over-treated with the vitrification solutions. Thus, Chen and Tian's cryopreservation method lacks general application to Japanese flounder embryos.

Channel-dependent permeation of water and glycerol in mouse morulae.

The cryosensitivity of mammalian embryos depends on the stage of development. Because permeability to water and cryoprotectants plays an important role in cryopreservation, it is plausible that the permeability is involved in the difference in the tolerance to cryopreservation among embryos at different developmental stages. In this study, we examined the permeability to water and glycerol of mouse oocytes and embryos, and tried to deduce the pathway for the movement of water and glycerol. The water permeability (L(P), microm min(-1) atm(-1)) of oocytes and four-cell embryos at 25 degrees C was low (0.63-0.70) and its Arrhenius activation energy (E(a), kcal/mol) was high (11.6-12.3), which implies that the water permeates through the plasma membrane by simple diffusion. On the other hand, the L(p) of morulae and blastocysts was quite high (3.6-4.5) and its E(a) was quite low (5.1-6.3), which implies that the water moves through water channels. Aquaporin inhibitors, phloretin and p-(chloromercuri) benzene-sulfonate, reduced the L(p) of morulae significantly but not that of oocytes. By immunocytochemical analysis, aquaporin 3, which transports not only water but also glycerol, was detected in the morulae but not in the oocytes. Accordingly, the glycerol permeability (P(GLY), x 10(-3) cm/min) of oocytes was also low (0.01) and its E(a) was remarkably high (41.6), whereas P(GLY) of morulae was quite high (4.63) and its E(a) was low (10.0). Aquaporin inhibitors reduced the P(GLY) of morulae significantly. In conclusion, water and glycerol appear to move across the plasma membrane mainly by simple diffusion in oocytes but by facilitated diffusion through water channel(s) including aquaporin 3 in morulae.

Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes.

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.

Water- and cryoprotectant-permeability of mature and immature oocytes in the medaka (Oryzias latipes).

The permeability of the plasma membrane plays a crucial role in the successful cryopreservation of oocytes/embryos. To identify a stage feasible for the cryopreservation of teleost oocytes, we investigated the permeability to water and various cryoprotectants of medaka (Oryzias latipes) oocytes at the germinal vesicle (GV) and metaphase II (MII) stages. In sucrose solutions, the volume changes were greater in GV oocytes than MII oocytes. Estimated values for osmotically inactive volume were 0.41 for GV oocytes and 0.74 for MII oocytes. Water-permeability (microm/min/atm) at 25 degrees C was higher in GV oocytes (0.13+/-0.01) than MII oocytes (0.06+/-0.01). The permeability of MII oocytes to various cryoprotectants (glycerol, propylene glycol, ethylene glycol, and DMSO) was quite low because the oocytes remained shrunken during 2 h of exposure in the cryoprotectant solutions at 25 degrees C. When the chorion of MII oocytes was removed, the volume change was not affected, except in DMSO solution, where dechorionated oocytes shrunk and then regained their volume slowly; the P(DMSO) value was estimated to be 0.14+/-0.01x10(-3) cm/min. On the other hand, the permeability of GV oocytes to cryoprotectants were markedly high, the P(s) values (x10(-3) cm/min) for propylene glycol, ethylene glycol, and DMSO being 2.21+/-0.29, 1.36+/-0.18, and 1.19+/-0.01, respectively. However, the permeability to glycerol was too low to be estimated, because GV oocytes remained shrunken after 2 h of exposure in glycerol solution. These results suggest that, during maturation, medaka oocytes become less permeable to water and to small neutral solutes, probably by acquiring resistance to hypotonic conditions before being spawned in fresh water. Since such changes would make it difficult to cryopreserve mature oocytes, immature oocytes would be more suitable for the cryopreservation of teleosts.

Permeability of mouse oocytes and embryos at various developmental stages to five cryoprotectants.

To assess the permeability of mouse oocytes and embryos, matured oocytes and embryos at various stages of development were placed in five cryoprotectant solutions at 25 C for 25 min. From the cross-sectional areas of the oocytes/embryos, the relative change in volume was analyzed. In oocytes, shrinkage was least extensive and recovery was quickest in the propylene glycol solution, showing that propylene glycol permeates the oocytes most rapidly. Dimethyl sulfoxide, acetamide, and ethylene glycol permeated the oocytes slightly more slowly than propylene glycol. The oocytes in glycerol shrunk extensively and then expanded marginally, indicating slow permeation. The volume changes of 1-cell and 2-cell embryos were similar to those of oocytes, showing little change in permeability. In 8-cell embryos, the volume recovered much faster than in the earlier stages especially in glycerol and acetamide. In morulae, the volume recovery was much faster in glycerol and in ethylene glycol; in ethylene glycol, the extent of shrinkage was small and the recovery was fast, indicating an extremely rapid permeation. Although the permeability of oocytes/embryos generally increased as embryo development proceeded, the degree of increase varied greatly among the cryoprotectants. Interestingly, the volume change in propylene glycol was virtually unaffected by the stage of development. Such information will be valuable for determining a suitable protocol for the cryopreservation of oocytes/embryos at different stages of development.