Over the last decades, advances in understanding basic phenomena in cryobiology has led the development of effective methods for the preservation of a very wide range of cells . However, the cryopreservation of cells with large volumes and multicellular systems and tissues, such as vertebrate oocytes and embryos, still remains a challenge in some cases [2–4]. Vertebrate oocytes show low surface area to volume ratio and low permeability to water and cryoprotectants, and as a consequence they are highly susceptible to formation of intracellular ice, cryoprotectant-toxicity, and osmotic stress. In addition, oocyte cryosurvival may change during development or as a result of different genotypes [4, 5].
One of the major obstacles with freezing of a cell is that intracellular water content crystallizes, leading to mechanical and physical damage [6, 7]. To limit the rate of ice crystals formation, the first step is to permeate cells with cryoprotectant agents, such as glycerol, propylene glycol, dimethyl sulfoxide or ethylene glycol, which should reach high intracellular concentrations, thus generating the driving force for the efflux of water by osmosis and of solutes by diffusion [6, 7]. This can be achieved either by a slow cooling rates with low concentration of cryoprotectants, or fast cooling after incubation in highly concentrated cryoprotectant solutions (vitrification) [8, 9]. An ideal cryoprotectant should thus exhibit low toxicity and rapidly permeate the cell, so that osmotic volume changes, that are a recognized form of cellular stress, are minimized [10–12]. Ethylene glycol shows the best efficiency/toxicity ratio on mammalian oocytes and embryos and is thus the most common selected cryoprotectant for slow-cooling or vitrification protocols [13–18]. However, the permeability of vertebrate oocytes and embryos to ethylene glycol is limited [19–24] and may change during development and differentiation [3, 4], resulting possibly in intracellular damage after cryopreservation [4, 16, 25].
It is now well established that biological membranes allow water to pass by simple diffusion through the lipid bilayer, or when rapid water permeability is required (in case of reabsorption, secretion or osmotic stress), through specialized membrane channels known as aquaporins. Aquaporins (AQPs) are integral membrane proteins present in all living organisms from bacteria to mammals . These proteins consist of six transmembrane helices connected by five loops, two of which bear the highly conserved asparagine-proline-alanine (NPA) motifs involved in the formation of the water pore . To date, 13 aquaporins have been identified in mammals (AQP0-12) and while most of them are water-selective, functional studies have identified a subgroup of channels (AQP3, -7, -9 and -10) that can transport also glycerol and urea, and are termed aquaglyceroporins (GLPs) [26, 27]. Some GLPs are also permeated by metalloids and a wide variety of noncharged solutes (e.g., carbamides, polyols, purines, pyrimidines), including cryoprotectants such as propylene glycol, ethylene glycol, acetamide and possibly dimethyl sulfoxide [28–32].
The discovery of aquaporins made it possible to use them for enhancing water and cryoprotectant permeability of cells [33–35]. In the baker's yeast (Saccharomyces cerevisiae), freeze tolerance during rapid freezing conditions positively correlates with high levels of expression of the water-selective aquaporins Aqy1 and Aqy2 [33, 36]. This positive effect has been explained by the rapid water efflux, especially at freezing temperatures, as a consequence of high levels of aquaporins in the plasma membrane of yeast cells, resulting in the reduction of intracellular ice crystal formation and cell damage. Similarly, heterologous expression of wild-type (WT) mammalian aquaporin-3 (AQP3) in vertebrate oocytes and embryos enhances both the efflux of water and influx of cryoprotectants into the cells [31, 34, 35, 37–41]. In the case of mouse oocytes, this method ameliorates oocyte tolerance to freezing and survival after thawing .
The increased permeability mediated by the artificial expression of AQP3 may be however insufficient for cryosurvival of cells or multicellular systems with large volumes, such as oocytes and embryos from lower vertebrates . This may be related to a decreased translation efficiency of AQP3 when expressed in an heterologous system and/or a limited water and solute permeability of AQP3 [39, 42, 43]. In addition, AQP3 is pH-dependant , and therefore, an effect of pH on AQP3-mediated water and cryoprotectant transport is likely to occur. This additional obstacle should be considered when using AQP3 in cryobiology as the intracellular pH is altered during volumetric changes in cells exposed to hypertonic or hypotonic environments [45–47]. Furthermore, the intracellular pH can be affected by the presence of cryoprotectants within the cell as some of them are dipolar aprotic or protic solvents and act as hydrogen bond acceptors or proton donors, and affect the acid-base equilibrium in mixed solvent systems . Therefore, exploiting further the possibilities of using aquaporins in cryobiology, it would be useful to develop aquaporins with enhanced permeability or pH insensitivity, by investigating GLPs of non-mammalian origin that may be more permeable to water and cryoprotectants than mammalian AQP3.
Recently, we have characterized the genomic repertoire of zebrafish (Danio rerio) aquaporins which contains seven GLP isoforms: DrAqp3a and -3b, DrAqp7, DrAqp9a and -9b, and DrAqp10a and -10b . Functional expression in Xenopus laevis oocytes demonstrated that all the zebrafish GLPs transport water, glycerol and urea in a similar manner as the mammalian orthologs , and therefore these channel proteins are of potential interest for cryobiology. In the present study, we have investigated both the pH sensitivity and cryoprotectant permeability of the zebrafish GLPs by using the X. laevis oocyte expression system. Interestingly, we found that both DrAqp3a and -3b isoforms are much more permeable to ethylene glycol than to glycerol or propylene glycol at neutral or alkaline pH, while all other zebrafish GLPs, as well as human AQP3, are more permeable to glycerol. Since ethylene glycol is a suitable cryoprotectant, we further engineered two DrAqp3 mutants that were insensitive to changes in pH or exhibit enhanced ethylene glycol permeability.