Equilibrium vitrification of oocytes using low concentrations of cryoprotectants.

In order to make dry ice transportation of vitrified embryos practical, a near-equilibrium vitrification was developed using a cryoprotectant solution (EDFS10/10a), by which mouse embryos at various stages were vitrified in a near-equilibrium environment. EDFS10/10a consisted of 10% (v/v) ethylene glycol, 10% (v/v) MeSO, 0.4 M sucrose and 24% (w/v) Ficoll PM70.

Quantification of residual cryoprotectants and cytotoxicity in thawed bovine ovarian tissues after slow freezing or vitrification.

Study Question: How much residual cryoprotectant remains in thawed/warmed ovarian tissues after slow freezing or vitrification?

Summary Answer: After thawing/warming, at least 60 min of diffusion washing in media was necessary to significantly reduce the residual cryoprotectants in ovarian tissues frozen by slow freezing or vitrification.

What Is Known Already: Ovarian tissue cryopreservation (OTC) by slow freezing has been the conventional method; while the vitrification method has gained popularity for its practicality. The main concern about vitrification is how much potentially toxic residual cryoprotectant remains in the warmed tissues at the time of transplantation.

Equilibrium vitrification of mouse embryos at various developmental stages using low concentrations of cryoprotectants.

We previously developed a new vitrification method (equilibrium vitrification) by which two-cell mouse embryos can be vitrified in liquid nitrogen in a highly dehydrated/concentrated state using low concentrations of cryoprotectants. In the present study, we examined whether this method is effective for mouse embryos at multiple developmental stages. Four-cell embryos, eight-cell embryos, morulae, and blastocysts were vitrified with EDFS10/10a, 10% (v/v) ethylene glycol and 10% (v/v) DMSO in FSa solution.

Equilibrium vitrification of mouse embryos using low concentrations of cryoprotectants.

Previously, we developed a method for vitrification of mouse embryos in a near-equilibrium state using EFS35c, PB1 medium containing 35% (v/v) ethylene glycol, and 0.98 M sucrose. This method has advantages in both slow freezing and vitrification.

Permeability of the plasma membrane to water and cryoprotectants in mammalian oocytes and embryos: Its relevance to vitrification.

The permeability of the plasma membrane to water and cryoprotectants is one of the important factors for determining the suitable condition for the vitrification of mammalian oocytes and embryos. Water and cryoprotectants move slowly through oocytes and early embryos, principally by simple diffusion, in the mouse, bovine, pig, and human. In contrast, water, glycerol, and ethylene glycerol move rapidly through morulae and blastocysts, principally by facilitated diffusion via aquaporin 3, in the mouse and bovine; whereas, in the pig, the permeability to water and these cryoprotectants increases not at the morula stage but at the blastocyst stage and further increases at the expanded blastocyst stage.

The movement of water and cryoprotectants across the plasma membrane of mammalian oocytes and embryos and its relevance to vitrification.

The permeability of the plasma membrane to water and cryoprotectants is one of the most important factors for determining suitable conditions for vitrification of mammalian oocytes and embryos. In mouse oocytes and early stage embryos, water and cryoprotectants move slowly, principally by simple diffusion. In contrast, in morulae (and probably blastocysts), water, glycerol, and ethylene glycerol move rapidly, principally by facilitated diffusion via aquaporin 3, and DMSO moves rapidly via channels other than aquaporin 3.

N, N-Dimethylglycine decreases oxidative stress and improves in vitro development of bovine embryos.

The antioxidant effect of N, N-dimethylglycine (DMG) on in vitro-produced (IVP) bovine embryos was examined. After in vitro fertilization, presumptive zygotes were cultured with or without 0.1 μM DMG under different oxygen tensions.

Rapid movement of water and cryoprotectants in pig expanded blastocysts via channel processes: its relevance to their higher tolerance to cryopreservation.

Pig oocytes and embryos are highly sensitive to cryopreservation; however, tolerance to cryopreservation increases in embryos at the expanded blastocyst stage. This increased tolerance may be attributed to a decrease in cytoplasmic lipid droplets at this stage. We previously showed that an increase in the permeability of the plasma membrane in mouse oocytes to water and cryoprotectants, caused by the artificial expression of aquaporin 3, an aquaglyceroporin, enhanced tolerance to cryopreservation.

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.

Equilibrium vitrification of mouse embryos at various developmental stages.

Previously, we developed a new method by which 2-cell mouse embryos can be vitrified in liquid nitrogen in a near-equilibrium state, and then kept at -80°C for several days. In the present study, we examined whether or not the method was effective for mouse embryos at other developmental stages. Eight-cell embryos, morulae, and expanded blastocysts of ICR mice were vitrified with ethylene glycol-based solutions, named EFSc because of their composition of ethylene glycol (30-40%, v/v) and FSc solution.

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.

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.

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.

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.

Equilibrium vitrification of mouse embryos.

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).

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).

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.

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.

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.

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.

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.

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.

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.

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.

Extra- and intra-cellular ice formation in Stage I and II Xenopus laevis oocytes.

We are currently investigating factors that influence intracellular ice formation (IIF) in mouse oocytes and oocytes of the frog Xenopus. A major reason for choosing these two species is that while their eggs normally do not possess aquaporin channels in their plasma membranes, these channels can be made to express. We wish to see whether IIF is affected by the presence of these channels.