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Laboratory of Animal Science,3 College of Agriculture, Kochi University, Nankoku, Kochi 783-8502, Japan
Frontier Science Research Center,4 University of Miyazaki, Kiyotake, Miyazaki 889-1692, Japan
ABSTRACT
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.
developmental biology,, embryo,, ovum
Since the first successful cryopreservation of mouse embryos in 1972 [1], various protocols have been developed to cryopreserve embryos and oocytes of many mammalian species. However, it is difficult to obtain high survival rates for embryos and oocytes at different stages using a single cryopreservation protocol. This may be related to differences in cryobiological properties among embryos and oocytes at different developmental stages. The permeability of the plasma membrane is an important factor in determining the tolerance of cells to cryopreservation, as the permeability modulates several major forms of cell injury caused by cryopreservation, i.e., damage from intracellular ice, cryoprotectant toxicity, and osmotic swelling [2].
In a preliminary study, we examined changes in the volumes of mouse oocytes and embryos in various cryoprotective solutions and showed that the pattern of permeation did not change from mature oocytes up to embryos at the two-cell stage, whereas it was drastically changed at around the morula stage [3]. The permeabilities to glycerol and ethylene glycol of embryos increased remarkably, and the permeabilities to acetamide and dimethylsulfoxide (DMSO) also increased slightly but significantly, whereas the permeability to propylene glycol did not change. This suggests that cryoprotectants permeate mouse embryos through different pathways in the plasma membrane, depending not only on the developmental stage of the cell but also on the cryoprotective agent itself.
Two pathways can be proposed for the movement of water and cryoprotectants across the plasma membrane. The first pathway involves simple diffusion across the plasma membrane, and the second pathway involves facilitated diffusion via channel processes. When water or a cryoprotectant moves by simple diffusion across the plasma membrane, the permeability to water (hydraulic conductivity, LP) or cryoprotectant (PS) should be quite low, whereas the Arrhenius activation energy (Ea) for permeability, which reflects the temperature dependence of permeability, should be high. On the other hand, when water and cryoprotectant permeate by facilitated diffusion through channel processes, the permeability should be high and the Ea value should be low. Verkman et al. [4] have suggested that an LP higher than 4.5 µm min–1 atm–1 and an Ea for the LP that is lower than 6 kcal mol–1 are suggestive of the movement of water principally through channels, and a low LP with an Ea for the LP higher than 10 kcal mol–1 is suggestive of movement principally via channel-independent diffusion. However, Verkman et al. [4] have pointed out that there appears to be no a priori theoretical basis for such values. Thus, the criteria would not be so strict but a rough estimation for deducing predominant pathways for water movement in the cell. Unfortunately, such criteria have not been reported for the movement of small neutral solutes across the plasma membrane, although it is reasonable to consider that a higher PS with a lower Ea for the PS is suggestive of the movement of solutes predominantly through channel processes, and a lower PS with a higher Ea for the PS is suggestive of movement predominantly via simple diffusion across the plasma membrane.
We have already shown in mouse oocytes at the metaphase II stage that water and glycerol move across the plasma membrane predominantly by simple diffusion, whereas, in morulae, they move predominantly by facilitated diffusion through water channel(s), including aquaporin (AQP) 3 [5]. There is no evidence that water-permeable channels other than AQPs expressed at a physiological level in the plasma membrane of the cell contribute to the overall permeability of the plasma membrane to water [6]. Therefore, AQP3 can be considered the major contributor to the movement of water in mouse morulae. It has also been shown that AQP3 can transport neutral solutes of lower molecular weight, such as glycerol [7]. Although AQP3 is permeable to cryoprotectants other than glycerol, such as ethylene glycol, propylene glycol, acetamide, and DMSO [8–12], it has not been directly shown that AQP3 expressed in mouse morulae contributes to the movement of such cryoprotectants or that other solute channels are also involved in this movement. It is important to know the mechanism by which these neutral solutes move across the plasma membrane of embryos so as to understand the cryobiological properties of embryos and the metabolism of small neutral solutes in embryos during the early stage of development.
The purpose of the present study was to clarify the pathways used for the movement of water and various cryoprotectants across the plasma membrane of mouse morulae, with special reference to the role of AQP3. Thus, we examined the Arrhenius activation energy for the permeability of mouse morulae to cryoprotectants and water in various cryoprotectant solutions, to determine whether cryoprotectants and water move across the plasma membrane by facilitated diffusion, as the movement of solutes through channels is less affected by temperature than movement through the lipid bilayer by simple diffusion. In addition, we examined the membrane permeability of mouse oocytes that artificially express AQP3 following the injection of the cRNA of Aqp3, as a model of mouse morulae that abundantly express AQP3. Finally, we examined the role of AQP3 and other solute channels in the movement of water and cryoprotectants in mouse morulae through the suppression of AQP3 expression by injecting the double-stranded RNA (dsRNA) of Aqp3.
Collection of Mouse Oocytes, Morulae, and Blastocysts
Mature female ICR mice (CLEA Japan, Inc., Tokoyo, Japan) were induced to superovulate with i.p. injections of 5 IU eCG and 5 IU hCG given 48 h apart. Ovulated unfertilized oocytes were collected from the ampullar portions of the oviducts 13 h after the injection of hCG, and were freed from cumulus cells by suspending them in PB1 that contained 37 units/ml hyaluronidase followed by washing with fresh PB1 (in vivo-matured oocytes). PB1 medium is modified PBS supplemented with glucose, pyruvate, penicillin, and BSA [13]. For the artificial expression of AQP3 in mouse oocytes, oocytes at the germinal vesicle stage (immature oocytes) were obtained by puncturing the follicles on the ovaries of female mice without injection of hCG, 46–50 h after the injection of eCG. These oocytes were injected with water or Aqp3 cRNA and cultured until they matured to the metaphase II stage (see below; injected oocytes). To obtain embryos, mature female ICR mice injected with eCG and hCG were mated with male ICR mice. Morulae that developed in vivo were collected from the uteri by flushing 76 h after the injection of hCG, and only compacted morulae were used (in vivo morulae). To prepare Aqp3 dsRNA inhected morulae, embryos at the 1-cell stage were collected by flushing the oviducts with PB1 medium 24 h after the injection of hCG, then injecting the embryos with water or dsRNA, and culturing for 56–68 h until they developed to the morulae stage (see below; injected morulae). In one experiment, injected embryos were cultured for 90 h, to allow them to develop to the blastocyst stage.
All experiments were approved by the Animal Ethics Committee of the College of Agriculture, Kochi University.
Measurements of Permeabilities to Cryoprotectants and Water of Mouse Oocytes and Embryos
The permeabilities to water and cryoprotectants of oocytes and embryos were determined by measuring the shrinkage and reswelling of the oocytes and embryos after transfer from isotonic PB1 medium to PB1 medium that contained either a cryoprotectant or 0.43 M sucrose at 15°C or 25°C for 5–10 min, as described previously [5, 14, 15]. The cryoprotectants used were 8% (vol/vol) ethylene glycol, 1.5 M acetamide, 9.5% (vol/vol) DMSO, 10% (vol/vol) propylene glycol, and 10% (vol/vol) glycerol. The concentrations of cryoprotectants were varied to prepare solutions with similar osmolalities. The osmolalities of ethylene glycol, propylene glycol, glycerol, and sucrose were calculated from the published data about colligative properties in aqueous solutions [16]. The osmolalities of acetamide and DMSO in aqueous solutions were measured with a vapor pressure osmometer (Vapro 5520; Wescor Inc., Logan, UT). The osmolality of PB1 medium (isotonic buffer) was measured with a freezing-point depression osmometer (OM801; Vogel, Giessen, Germany). The total osmolality of each solution used is shown in Table 1.
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Each oocyte or embryo was placed in a 100-µl drop of PB1 medium and covered with paraffin oil in a Petri dish (90 x 10 mm), and was held with a holding pipette (outer diameter of 80–120 µm) that was connected to a micromanipulator on an inverted microscope. The inner diameter of the holding pipette was small enough so as not to distort the oocyte or embryo. The temperature of the paraffin oil covering the various solutions was considered to be the temperature of the solution, and was maintained at 15 ± 1°C or 25 ± 1°C by controlling the temperature of the room. The oocyte or embryo held with the holding pipette was then covered with a covering pipette that had a larger inner diameter (approximately 200 µm) and that was connected to another micromanipulator. Then, by sliding the dish, the oocyte or embryo was introduced into a drop of PB1 medium that contained sucrose or cryoprotectant. By removing the covering pipette, the oocyte or embryo was abruptly exposed to the solution [3, 5, 15]. The microscopic images of the oocytes and embryos during exposure to the solution were recorded at 0.5-sec intervals with a time-lapse videotape recorder (ETV-820; Sony, Tokyo, Japan). The cross-sectional areas of the oocytes and embryos were measured using an image analyzer (VM-50; Olympus, Tokyo, Japan) and the results are expressed as the relative cross-sectional area, S, calculated by dividing the cross-sectional area by the area of the same oocyte and embryo in isotonic PB1 medium. The relative volume was obtained from: 
. The permeabilities to water and cryoprotectants of the oocytes and embryos were determined by fitting the movement of water and cryoprotectants using a two-parameter formalism, as described previously [5, 14, 15]. The related constants and parameters used are listed in Table 2.
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For in vivo-matured oocytes, the volume change was measured for 10 min at 15°C and 25°C independently, using different oocytes, since the oocytes shrunk and reswelled very slowly in the cryoprotectant solutions, especially at 15°C. Thus, the Ea of Ps and LP was obtained from the Arrhenius plots of data from different oocytes at the two temperatures.
For the in vivo morulae, the volume change was measured in PB1 medium that contained cryoprotectant at 15°C for 5 min. Then, the morula was transferred to PB1 medium that contained 0.5 M sucrose at 25°C, retained for 20 min, in order to remove the intracellular cryoprotectant, and then equilibrated with isotonic PB1 medium at 25°C for at least 10 min. Then, the volume changes of the morulae in the same solution were measured at 25°C for 5 min. Thus, the Ea of Ps and LP was obtained from Arrhenius plots of the data from the same morula at the two temperatures.
Measurements of Permeabilities to Water and Cryoprotectants of Mouse Oocytes Injected with the cRNA of Aqp3
To examine the contribution of AQP3 to the movement of water and cryoprotectants in mouse morulae, we measured the PS and LP of mouse oocytes that artificially expressed mouse AQP3. This is a model for mammalian cells that express AQP3 abundantly, such as mouse morulae, since AQP3 is scarcely expressed in mouse oocytes [5]. Mouse Aqp3 cRNA was synthesized by essentially the same method as described elsewhere for the synthesis of rat Aqp3 cRNA [11, 15]. Briefly, mouse Aqp3 cDNA was cloned from mouse kidney cDNA by PCR with the sense primer 5'-CGAATTCGTCTCGGGTGCTTGCGCT-3' and antisense primer 5'-CTCTAGAGACACAGGGAGCGGTTTAG-3' (underlined sequences indicate inserted EcoRI and XbaI sites, respectively). These primers were derived from the mouse Aqp3 sequence [18] (GenBank accession no. AF104416). The PCR cycle had the following profile: 30 cycles of 94°C for 30 sec, 57°C for 30 sec, and 72°C for 60 sec. The PCR product contained the open reading frame (ORF) of mouse Aqp3. The EcoRI/XbaI fragment of the PCR product was subcloned into the EcoRI/XbaI site of the pTnT Vector (Promega, Madison, WI), which is an in vitro-transcription/translation plasmid. After digestion of the construct with BamHI, the capped cRNA of Aqp3 was synthesized using SP6 polymerase (Takara Shuzo, Otsu, Japan). The BamHI, EcoRI, and XbaI restriction enzymes were obtained from Toyobo (Osaka, Japan).
Immature mouse oocytes were injected with the cRNA of mouse Aqp3 as described in our previous study [15], in which they were injected with the cRNA of rat Aqp3. Briefly, an immature oocyte was held with a holding pipette connected to a micromanipulator on an inverted microscope and injected with 2–10 pl of water (control) or Aqp3 cRNA solution (1 pg/pl) with an injection needle connected to another micromanipulator. As an additional control, noninjected oocytes were used. Noninjected, water-injected, and cRNA-injected oocytes were cultured in a humidified CO2 incubator (5% CO2 and 95% air) at 37°C for 12–15 h in MEM that was supplemented with 10% fetal calf serum, 50 µg/ml sodium pyruvate, 2 mM glutamine, 60 µg/ml penicillin G, and 50 µg/ml streptomycin. Only oocytes that had a polar body after being cultured were considered to have matured normally and were used in the experiments. Only a limited number of oocytes was used in each replicate experiment as the period of culture for expression of AQP3 was limited to 12–15 h.
The permeabilities to water and cryoprotectants of the oocytes were measured at 25°C. Initially, the LP values of noninjected, water-injected, and cRNA-injected oocytes were determined by measuring the shrinkage of oocytes for 5 min after their transfer from isotonic PB1 medium to PB1 medium that contained 0.43 M sucrose at 25°C. The oocytes were transferred to isotonic PB1 medium and equilibrated at 25°C for 30 min. Then, the oocytes were transferred to PB1 medium that contained 10% (vol/vol) glycerol, 8% (vol/vol) ethylene glycol, 1.5 M acetamide, 9.5% (vol/vol) DMSO or 10% (vol/vol) propylene glycol, and their volume changes were measured at 25°C for 5 min.
Measurements of the Permeabilities to Water and Cryoprotectants of Mouse Morulae and Blastocysts Injected with Aqp3 dsRNA
To study the role of AQP3 in the movement of water and cryoprotectants in mouse morulae, we measured the Ps and LP of morulae in which the expression of AQP3 was suppressed by the dsRNA of mouse Aqp3.
The cDNA of mouse Aqp3 was cloned from mouse kidney cDNA by PCR with the sense primer 5'-TAATACGACTCACTATAGGGGTCTCGGGTGCTTGCGCTA-3' and antisense primer 5'-TAATACGACTCACTATAGGGGACACAGGGAGCGGTTTA-3' (underlined are the T7 promoter sequences). These primers were derived from the mouse Aqp3 sequence [18]. The PCR was conducted for 35 cycles of 94°C for 30 sec, 62°C for 30 sec, and 72°C for 30 sec. The PCR product contained the ORF of Aqp3. From this product, the dsRNA of Aqp3 was synthesized with the T7 RiboMAX Express RNAi System (Promega), according to the manufacturer's instructions.
An embryo at the 1-cell stage was held with a holding pipette connected to a micromanipulator on an inverted microscope in PB1 medium and injected with 2–10 pl of water (control) or the Aqp3 dsRNA solution (1 pg/pl) with an injection needle connected to another micromanipulator. As an additional control, noninjected embryos were used. The noninjected embryos and the injected embryos with normal shape just after injection were cultured in M16 medium that was supplemented with 10 µM EDTA, 1 mM glutamine, and 10 µM ß-mercaptoethanol [19] in a humidified CO2 incubator at 37°C (5% CO2 and 95% air) for 56–68 h, until they developed to the morula stage; only compacted morulae were used in the experiments. The morulae derived from noninjected embryos and the morulae derived from embryos injected with water or Aqp3 dsRNA are referred to as noninjected morulae and water-injected or dsRNA-injected morulae, respectively. No significant differences were noted in terms of the ability of 1-cell embryos to develop to the morulae stage between the noninjected and injected embryos of normal shape and color just after injection (data not shown). We also used in vivo-matured oocytes as a control. In one experiment, dsRNA-injected 1-cell embryos were cultured for 90 h to obtain dsRNA-injected blastocysts.
The volume changes of the morulae and oocytes were measured in PB1 medium that contained 0.43 M sucrose at 25°C for 5 min to determine the LP. The morulae and oocytes were equilibrated in isotonic PB1 medium at 25°C for 30 min. Then, they were transferred to PB1 medium that contained 10% (vol/vol) glycerol, 8% (vol/vol) ethylene glycol, 1.5 M acetamide, 9.5% (vol/vol) DMSO or 10% (vol/vol) propylene glycol, and their volume changes were measured at 25°C for 10 min, except for the noninjected and water-injected morulae in glycerol, acetamide, and DMSO solutions; in these cases, the morulae were exposed to the solutions at 25°C for 5 min. For blastocysts, the blastocoel was punctured with a fine-gauge needle and the LP was determined in the sucrose solution at 25°C for 5 min.
The dsRNA of Aqp3 used in the present study includes the ORF of mouse Aqp3 and therefore, it includes the consensus sequences of the Aqp family. Thus, it is possible that the difference in membrane permeability observed between noninjected or water-injected morulae and dsRNA-injected morulae was derived not only from the suppression of expression of AQP3 but also from the suppression of expression of other AQPs. However, we have shown previously that only the expression of mRNAs for Aqp3 and Aqp7 can be detected in the morulae of the ICR mouse that we used [20], and the morulae express AQP3 protein abundantly but not AQP7 protein [5]. Barcroft et al. [21] have also shown marked expression of AQP3 in the morulae of another mouse strain. Therefore, the difference between noninjected morulae (and water-injected morulae) and dsRNA-injected morulae seen in the present study is mainly attributable to the suppression of AQP3.
Immunological Detection of AQP3 in Mouse Morulae Injected with Aqp3 dsRNA
To detect the expression of AQP3, we used a commercially available anti-rat AQP3 rabbit antibody that cross-reacts with mouse AQP3. Murine noninjected and dsRNA-injected morulae and in vivo-matured oocytes were fixed with a 2% paraformaldehyde solution that contained 0.01 M sodium metaperiodate, 0.075 M lysine, and 0.075 M phosphate buffer (pH 7.4) at 4°C for 60 min. After washing with PBS that contained 5 mg/ml BSA, the cells were permeabilized with PBS that contained 0.25% Triton X-100. They were then incubated with a blocking solution, PBS that contained 5% non-immune goat serum (Biosource, Camarillo, CA) and 5 mg/ml BSA, at 25°C for 60 min. After rinsing, the cells were incubated with anti-rat AQP3 rabbit antibody (1:50 dilution) (Chemicon International Inc., Temecula, CA) in blocking solution at 25°C for 60 min. After rinsing, the oocytes and morulae were incubated with diluted fluorescein isothiocyanate (FITC) -conjugated anti-rabbit immunoglobulin goat antibody (1:400) (MP Biomedicals LLC-Cappel Products, Solon, CA) in blocking solution at 25°C for 30 min, rinsed again, and observed under a fluorescence microscope.
The PS and LP values of the oocytes and morulae were compared using the Student t-test (P < 0.05 was considered significant).
Permeabilities of Mouse Morulae to Cryoprotectants and Water and the Arrhenius Activation Energies in Cryoprotectant Solutions
In order to assess the involvement of channel processes in the movement of various cryoprotectants and water in mouse morulae, we first calculated the Ea for the PS and LP of in vivo morulae, together with those of in vivo-matured oocytes as a control, from the volume changes in cryoprotectant solutions.
In general, when cells are suspended in a hypertonic solution that contains a permeating cryoprotectant, they are expected to shrink and then regain their volume rapidly if their LP and PS values are high, whereas they are expected to shrink and regain volume slowly if their LP and PS values are low. On the other hand, if the LP is low and the PS is high, the cells are expected to slowly shrink a little but rapidly regain their volume, and if the LP is high and the PS is low, the cells are expected to shrink rapidly but slowly regain their volume.
Figure 1 shows the changes in volume of in vivo-matured oocytes and in vivo morulae in cryoprotectant solutions at 15°C and 25°C. The matured oocytes shrunk slowly and regained volume slowly at 25°C in all the cryoprotectant solutions (Fig. 1, A–D). Shrinkage and reswelling were much slower at 15°C than at 25°C. On the other hand, the changes in volume of the in vivo morulae differed among the cryoprotectant solutions. In the ethylene glycol solution, the morulae shrunk little and reswelled quickly at 15°C and 25°C (Fig. 1A). The volume change at 15°C was slightly slower than that at 25°C. In the acetamide and DMSO solutions, the morulae shrunk very rapidly at both 15°C and 25°C, and regained their volume relatively rapidly, albeit slightly slower at 15°C than at 25°C (Fig. 1, B and C). In the propylene glycol solution, the in vivo morulae shrunk slowly and regained their volume slowly, like the in vivo-matured oocytes, and the volume change was much slower at 15°C than at 25°C (Fig. 1D).
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Tables 3 and 4 show the PS and LP, respectively, of in vivo-matured oocytes and in vivo morulae in cryoprotectant solutions at 15°C and 25°C, as calculated from the data shown in Figure 1, and the Ea for the PS and LP calculated from the values. The permeability to ethylene glycol (PEG) of in vivo-matured oocytes was low and markedly different at 15°C from that at 25°C. Thus, the Ea for the PEG of the oocytes was high (17.3 kcal/mol) (Table 3). On the other hand, the PEG values of the in vivo morulae at 15°C and 25°C were high, and the Ea for the PEG was low (9.1 kcal/mol). In the acetamide and DMSO solutions, similar differences in the permeabilities to acetamide (PAA) and DMSO (PDMSO) and the Ea for the PS of in vivo-matured oocytes and in vivo morulae were observed (Table 3). Thus, as previously observed for the movement of glycerol [5], channel processes appear to be involved in the movement of ethylene glycol, acetamide, and DMSO in mouse morulae. On the other hand, the Ea values for the permeability to propylene glycol (PPG) of in vivo-matured oocytes and in vivo morulae were high and almost identical (20.3 kcal/mol). Thus, most propylene glycol molecules appear to permeate in vivo morulae not by facilitated diffusion but by simple diffusion across the plasma membrane.
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The LP of in vivo-matured oocytes was low in all cryoprotectant solutions examined and the Ea for the LP was also high (9.5–14.6 kcal/mol) (Table 4), which suggests that most of the water molecules move across the plasma membrane of mouse oocytes by simple diffusion. On the other hand, the LP and the Ea values for LP of in vivo morulae were markedly different among the cryoprotectant solutions. The LP of in vivo morulae in DMSO solution at 25° C was high and the Ea for the LP was low (6.9 kcal/mol), which suggests that water moves efficiently by facilitated diffusion through water channels. The LP of the acetamide solution at 25° C was also high and the Ea for the LP was also low (8.6 kcal/mol), which suggests that many water molecules move by facilitated diffusion through water channels in the in vivo morulae. On the other hand, the LP values in ethylene glycol and propylene glycol solutions at 25°C were low (0.53–0.97 µm min–1 atm–1) and the Ea values for the LP were high (13.5–15.1 kcal mol–1), which suggests that in the presence of these cryoprotectants, most of the water molecules do not move by channel processes but by simple diffusion across the plasma membrane of in vivo morulae, although AQP3 is expressed abundantly in these cells [5, 21]. Using our previous data on the changes in volume of mouse oocytes and embryos in 10% (v/v) glycerol solution at 15°C and 25°C [5], we recalculated the Ea for LP of in vivo-matured oocytes and in vivo morulae in 10% (vol/vol) glycerol at 15°C and 25°C. The Ea for LP of in vivo morulae in the glycerol solution (9.1 kcal/mol) was lower than that of in vivo-matured oocytes (13.5 kcal/mol).
Thus, many water molecules move efficiently across the plasma membrane of mouse morulae via water channels in acetamide and DMSO solutions, as well as in the glycerol solution, although this movement is suppressed in the ethylene glycol and propylene glycol solutions.
Permeabilities to Water and Cryoprotectants of Mouse Oocytes Artificially Expressing Mouse AQP3
Figure 2 shows the changes in volume of noninjected oocytes and oocytes injected with water or murine Aqp3 cRNA in a sucrose solution and in the cryoprotectant solutions. In the sucrose solution (Fig. 2A), cRNA-injected oocytes shrunk rapidly, which indicates that water moves efficiently through the AQP3 expressed in the oocytes injected with Aqp3 cRNA. The volume change of the cRNA-injected oocytes was similar to that of the in vivo morulae in our previous study [5]. The LP was remarkably higher than that of the noninjected or water-injected oocytes (Table 5). These results indicate that mouse AQP3 is expressed abundantly in cRNA-injected oocytes, as in morulae.
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In the glycerol solution, the cRNA-injected oocytes shrunk and regained their volume rapidly, whereas noninjected and water-injected oocytes shrunk slowly and regained their volume very slowly (Fig. 2B). The volume change of the cRNA-injected oocytes was similar to that of the in vivo morulae observed in our previous study [5]. The LP and the permeability to glycerol (PGly) of the cRNA-injected oocytes were markedly higher than those of noninjected and water-injected oocytes (Tables 6 and 7). Thus, in the glycerol solution, water and glycerol move efficiently through AQP3.
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In the ethylene glycol solution, the cRNA-injected oocytes shrunk only slightly and swelled thereafter, whereas the noninjected and water-injected oocytes shrunk slowly and regained their volumes very slowly (Fig. 2C). The volume change of the cRNA-injected oocytes was similar to that of in vivo morulae (Fig. 1A). The PEG of the cRNA-injected oocytes was markedly higher than that of noninjected or water-injected oocytes (Table 6), whereas the LP of the cRNA-injected oocytes (0.31 µm min–1 atm–1) was low and was not significantly different from that of noninjected or water-injected oocytes (0.52–0.54 µm min–1 atm–1) (Table 7). These results are quite similar to those obtained for the in vivo morulae (Tables 3 and 4). Thus, while ethylene glycol permeates AQP3 efficiently, water shows little movement through AQP3.
In the acetamide and DMSO solutions, the cRNA-injected oocytes shrunk very rapidly but regained their volume slowly, whereas water-injected oocytes shrunk and regained their volume slowly (Fig. 2, D and E). The slow reswelling was different from the reswelling of in vivo morulae (Fig. 1, B and C). The LP of the cRNA-injected oocytes in the solutions markedly increased from that of the noninjected or water-injected oocytes (Table 7), whereas the PAA and PDMSO of the cRNA-injected oocytes were low and not significantly different from those of the noninjected and water-injected oocytes (Table 6). The permeability of AQP3-expressing oocytes to these cryoprotectants was substantially lower than that of in vivo morulae (Table 3). These results show that in the presence of acetamide and DMSO, water moves efficiently through AQP3 but these cryoprotectants exhibit poor permeation through AQP3.
In the propylene glycol solution, the cRNA-injected oocytes shrunk slowly and regained their volume slowly, similar to the noninjected oocytes (Fig. 2F). The volume change was similar to that of the in vivo morulae (Fig. 1D). The PPG and LP of the cRNA-injected oocytes were 3.38 x 10–3 cm min–1 and 1.67 µm min–1 atm–1, respectively (Tables 6 and 7). The PPG was not significantly higher than that of noninjected or water-injected oocytes (1.99–2.23 x 10–3 cm/min) (Table 6), and the LP was slightly higher than that of noninjected or water-injected oocytes (0.55–0.74 µm min–1 atm–1) (Table 7). These characteristics were similar to those of in vivo morulae (Tables 3 and 4). These results suggest that in the presence of propylene glycol, little water or propylene glycol moves through AQP3.
Permeabilities to Water and Cryoprotectants of Mouse Morulae Injected with Double-Stranded RNA of Mouse Aqp3
Figure 3 shows that the expression of AQP3 in morulae was suppressed by injecting the dsRNA of Aqp3. The anti-AQP3 antibody detected marked expression of AQP3 in a noninjected morula (Fig. 3A), whereas expression was scarcely detected in a dsRNA-injected morula (Fig. 3B) or vivo-matured oocyte (Fig. 3C). Thus, the injection of Aqp3 dsRNA into embryos at the 1-cell stage strongly suppresses the expression of AQP3 at the morula stage. We used these morulae for the following experiments.
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In the hypertonic sucrose solution, noninjected and water-injected morulae shrunk very rapidly at 25°C (Fig. 4A), as did the in vivo morulae in our previous study [5], whereas dsRNA-injected morulae shrunk slowly, similar to the vivo-matured oocytes (Fig. 4A). The LP of the water-injected morulae in the sucrose solution was substantially high, similar to that of noninjected morulae (Table 8), whereas the value for dsRNA-injected morulae was quite low and close to that of in vivo-matured oocytes. However, the LP value of the dsRNA-injected morulae (1.14 ± 0.85 µm min–1 atm–1) was slightly higher and the standard deviation was larger than that of in vivo-matured oocytes (0.68 ± 0.12 µm min–1 atm–1) (Table 8). Thus, some of the dsRNA-injected morulae that we used may have retained some expression of the AQP3 protein. In order to examine the duration of silencing AQP expression in embryos by the injection of Aqp3 dsRNA, we examined the LP of dsRNA-injected blastocysts. As for the morulae, the LP value was low (1.11 ± 0.19 µm min–1 atm–1) (Table 8). These results show that most of the facilitated diffusion in mouse morulae (and blastocysts) relies on AQP3.
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In the glycerol solution, the noninjected and water-injected morulae shrunk rapidly and regained their volume rapidly (Fig. 4B), as did the in vivo morulae in our previous study [5]. On the other hand, dsRNA-injected morulae shrunk slowly and regained their volume slowly, albeit faster than oocytes. The PGly values of noninjected and water-injected morulae were very high (3.14–3.67 x 10–3 cm/min), whereas that of dsRNA-injected morulae was significantly low (1.29 x 10–3 cm/min) (Table 9), although the value for the dsRNA-injected morulae was significantly higher than that for the in vivo-matured oocytes (0.01 x 10–3 cm min–1). The LP values of noninjected and water-injected morulae in the glycerol solution were also high (2.17–2.21 µm min–1 atm–1), whereas that of dsRNA-injected morulae (0.64 µm min–1 atm–1) was markedly low, similar to the value for in vivo-matured oocytes (0.59 µm min–1 atm–1) (Table 10). This suggests that, in the presence of glycerol, water and glycerol move efficiently through AQP3 in mouse morulae.
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In the ethylene glycol solution, noninjected and water-injected morulae shrunk slightly and regained their volume rapidly (Fig. 4C). On the other hand, as in the glycerol solution, dsRNA-injected morulae shrunk slowly (like the in vivo-matured oocytes) and regained their volume slowly, albeit faster than the oocytes. The PEG values of noninjected and water-injected morulae were substantially high (8.48–11.08 x 10–3 cm/min), whereas that of dsRNA-injected morulae (2.35 x 10–3 cm/min) was significantly lower (Table 9). However, the value was significantly higher than that of in vivo-matured oocytes (0.58 x 10–3 cm/min). On the other hand, the LP values of noninjected and water-injected morulae in the ethylene glycol solution were as low (0.43–0.44 µm min–1 atm–1) as that of in vivo oocytes (0.42 µm min–1 atm–1) (Table 10). The LP of dsRNA-injected morulae was also low (0.50 µm min–1 atm–1). This suggests that, in the presence of ethylene glycol, water molecules show little movement through AQP3, whereas ethylene glycol permeates rapidly through AQP3 of mouse morulae.
In the acetamide and DMSO solutions, noninjected and water-injected morulae shrunk and regained their volume rapidly, whereas in vivo-matured oocytes shrunk and regained their volume slowly (Fig. 4, D and E). On the other hand, dsRNA-injected morulae shrunk slowly (like the in vivo-matured oocytes) but regained their volume rapidly (like the noninjected morulae). The PAA and PDMSO of dsRNA-injected morulae were high (4.13 and 2.36 x 10–3 cm/min, respectively), as were those of noninjected and water-injected morulae (2.79–3.43 and 1.86–1.92 x 10–3 cm/min, respectively), whereas those of in vivo-matured oocytes were low (0.90–1.00 x 10–3 cm/min) (Table 9). On the other hand, the LP values of noninjected and water-injected morulae in the acetamide and DMSO solutions were high (1.38–1.62 and 1.59–2.16 µm min–1 atm–1, respectively), whereas the LP of dsRNA-injected morulae was low (0.71–0.81 µm min–1 atm–1), as in the case of in vivo-matured oocytes (0.47–0.49 µm min–1 atm–1) (Table 10). This suggests that in the presence of acetamide or DMSO, many water molecules move through the AQP3 of mouse morulae, whereas acetamide and DMSO molecules permeate very poorly through the AQP3 of mouse morulae. Facilitated diffusion pathways other than AQP3 appear to be involved in the movement of acetamide and DMSO.
In the propylene glycol solution, noninjected and water-injected morulae shrunk and regained their volumes slowly (Fig. 4F). Although they regained their volumes slightly faster than in vivo-matured oocytes, the volume changes were essentially similar to that of the oocytes. The volume change of dsRNA-injected morulae was quite similar to that of noninjected or water-injected morulae. The PPG values of noninjected and water-injected morulae (2.68–3.29 x 10–3 cm/min) were quite similar to that of dsRNA-injected morulae (2.67 x 10–3 cm/min), and the value was slightly but significantly higher than that of in vivo-matured oocytes (1.68 x 10–3 cm/min) (Table 9). The LP values of noninjected and water-injected morulae in the propylene glycol solution were low (0.79–0.93 µm min–1 atm–1) (Table 10). The LP of dsRNA-injected morulae was also low (0.68 µm min–1 atm–1), similar to that of the matured oocytes (0.48 µm min–1 atm–1) (Table 10). These results suggest that in the presence of propylene glycol, few water or propylene glycol molecules move through the AQP3 of mouse morulae.
In the present study, we deduced the mechanism by which water and various cryoprotectants move across the plasma membrane of mouse morulae, and clarified the role of AQP3 in this movement.
In mouse oocytes, water and the cryoprotectants examined in the present study move very slowly across the plasma membrane and the Ea for the LP and PS was high (Tables 3, 4 , 9, and 10), which suggests that water and cryoprotectants move across the plasma membrane of mouse oocytes principally by simple diffusion across the plasma membrane. On the other hand, in general, water and cryoprotectants move rapidly across the plasma membrane of mouse morulae in cryoprotectant solutions, and the Ea values for LP and PS values were relatively low, except for those measured in the propylene glycol solution (Tables 3 and 4, and [5]), which suggests that water and most cryoprotectants move across the plasma membrane of mouse morulae via channel-mediated processes. However, the contributions of channel processes to movement differed greatly among the cryoprotectants in the solutions.
Without cell-permeating solutes, most water molecules move across the plasma membrane of mouse morulae through AQP3, since in the sucrose solution, the LP of noninjected morulae was substantially high and the LP of AQP3 dsRNA-injected morulae was low and close to that of in vivo-matured oocytes (Table 8). Therefore, AQP3 must be the major contributor to the movement of water in mouse morulae, as speculated in our previous study [5]. In addition, the LP of Aqp3 dsRNA-injected blastocysts was quite low, suggesting that Aqp3 dsRNA injected in embryos retains its suppressive effect even 90 h after injection.
In the glycerol solution, most of the water and glycerol molecules appeared to move through AQP3 in morulae, as the LP and PGly values of the morulae were much higher than those of the oocytes (Tables 9 and 10, and [5]), the Ea values for the LP and PGly were low [5], and the high LP and PGly values markedly decreased with the injection of dsRNA (Tables 9 and 10). This speculation is supported by the finding that the membranes of noninjected morulae (Tables 9 and 10) had similar properties to those of oocytes that artificially expressed AQP3 (Tables 6 and 7). The PGly value of dsRNA-injected morulae was significantly lower than that of noninjected morulae but was significantly higher than that of oocytes (Table 9). Since the LP of the dsRNA-injected morulae in the glycerol solution was as low as that of in vivo-matured oocytes (Table 10), the expression level of AQP3 protein appears to be low in dsRNA-injected morulae. Although it is possible that weak expression of AQP3 in the dsRNA-injected morulae contributed to the movement of glycerol, it is also possible that channels other than AQP3 are involved in the movement. Further studies are needed to clarify this point.
In the ethylene glycol solution, most of the ethylene glycol molecules appeared to permeate the morulae through AQP3, since the PEG of the in vivo morulae was much higher than that of the in vivo-matured oocytes (Table 3), the Ea for the PEG was low (Table 3), and the high PEG value markedly deceased following the injection of Aqp3 dsRNA (Table 9). However, little water moved through AQP3, as the LP values of the in vivo morulae and noninjected morulae were as low as those of the oocytes or dsRNA-injected morulae (Tables 4 and 10). Similar properties were observed for AQP3-expressing oocytes (Tables 6 and 7). It seems likely that in the ethylene glycol solution, rapid permeation of ethylene glycol into the morulae through AQP3 would decrease the movement of water to the outside of the morulae through AQP3, since both ethylene glycol molecules and water molecules would move through the same channel, AQP3. Thus, the osmotic difference between the outside and inside of the plasma membrane of morulae would be lost in a very short period by the rapid permeation of ethylene glycol into the morulae. Further studies are needed for clarify the mechanism of this phenomenon. Similar to PGly, the PEG of dsRNA-injected morulae was significantly lower than that of noninjected morulae but was significantly higher than that of oocytes (Table 9). Therefore, it is possible that channels other than AQP3 are also involved in the movement of ethylene glycol in mouse morulae.
In the acetamide and DMSO solutions, the PAA and PDMSO of the dsRNA-injected morulae were high and similar to those of noninjected morulae (Table 9), although the LP of the dsRNA-injected morulae was markedly low (Table 10). Since the PAA and PDMSO of in vivo morulae were higher than those of in vivo oocytes and the Ea values for the PAA and PDMSO of in vivo morulae were much lower that those of oocytes (Table 3), most of the acetamide and DMSO molecules appear to permeate mouse morulae via channel processes. Therefore, acetamide and DMSO may permeate mouse morulae not through AQP3 but through other channels, whereas most water molecules permeate the morulae through AQP3 in acetamide and DMSO solutions. This speculation was supported by the findings that in AQP3-expressing mouse oocytes, the PAA and PDMSO did not increase but the LP increased markedly (Tables 6 and 7). It has been shown in AQP3-expressing Xenopus oocytes that acetamide can permeate AQP3 effectively [8, 9, 12]. Very recently, we have shown that DMSO can permeate AQP3 expressed in Xenopus and fish oocytes [11, 12]. Just why the properties of artificially expressed AQP3 in oocytes differ among vertebrate species is not clear. Sidoux-Walter et al. [22] have reported that urea transporter 3, which is another neutral solute channel, transports water and various solutes in Xenopus oocytes when it is expressed in the oocyte at a much higher than physiological level, whereas it transports only urea when expressed at the physiological level. Thus, it is possible that the over-expression of AQP3 in frog and fish oocytes causes abnormal permeation by solutes, and that AQP3 does not transport acetamide and DMSO at the physiological expression level. Further studies are needed to clarify this discrepancy.
In the propylene glycol solution, the permeabilities to water and propylene glycol of noninjected morulae were similar to those of dsRNA-injected morulae (Tables 9 and 10), although it has been shown that propylene glycol permeates AQP3 effectively in frog and fish oocytes [9–12]. Since the Ea values for PPG and LP of in vivo morulae in the propylene glycol solution were quite high (Tables 3 and 4), the movement of water through AQP3 must affect the movement of propylene glycol, and little water or propylene glycol moves through AQP3. This speculation is supported by our present results that the permeability of AQP3-expressing mouse oocytes in the propylene glycol solution (Tables 6 and 7) was quite similar to those of noninjected morulae and dsRNA-injected morulae and close to that of in vivo-matured oocytes (Tables 9 and 10). Further studies are needed to clarify this discrepancy with respect to the properties of AQP3-expressing oocytes among the mouse, frog, and fish.
In the present study, we show that AQP3 expressed in mouse morulae plays important roles in the movement of water and neutral solutes but that this contribution depends on the solutes. Since we have already shown that the membrane permeability of mouse embryos is similar to that of bovine embryos [23], the permeability-related properties of embryos may be similar among different species of mammals. If so, the results of the present study provide important information on the cryobiological properties of embryos in many mammalian species and for the formulation of cryopreservation protocols for these embryos.
We also suggest that solute channels other than AQP3 are involved in the facilitated diffusion of some cryoprotectants, e.g., acetamide and DMSO. Ma et al. [18] have reported that Aqp3 knockout mice develop to term and are grossly normal, except for the polyurea. Thus, it is possible that in Aqp3 knockout mouse morulae, neutral solute channels other than AQP3 transport various solutes if AQP3 plays an important role in embryos after the morula stage, and therefore, the morulae can develop to term. Further studies are needed to understand the importance of the transport of small neutral solutes in the development of mammalian embryos.
FOOTNOTES
1Supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology; and the Ministry of Health, Labour and Welfare of Japan. 
Correspondence: 2Correspondence. FAX: 81 88 864 5200; e-mail: keisuke{at}kochi-u.ac.jp
Received: 4 December 2006.
First decision: 27 December 2006.
Accepted: 9 April 2007.
REFERENCES
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