Animals

Adult Japanese tree frogs, H. japonica, of both sexes were supplied by Oh-uchi Aquatic Animal Supply (Saitama, Japan). The frogs used in this study had a snout-vent length of approximately 3.0 cm and weighed approximately 2.8 g. For freezing experiments, non-hibernating frogs that had been captured in late November were kept in plastic containers with moistened moss, fed a diet of mealworms, and exposed to natural conditions of temperature and light cycle. The frogs hibernated from December to February and were used for the freezing experiment. Active tree frogs that were captured in April were kept under normal laboratory conditions and were fed mealworms for one week before sampling. All animal experiments were carried out in compliance with the Guide for Care and Use of Laboratory Animals of Shizuoka University.

Molecular cloning of AQP-h9 cDNA

Total RNA was extracted from the frog liver with TRIzol reagent (Life Technologies, Carlsbad, CA, USA). A total of 3 µg RNA was reverse transcribed at 37°C for 50 min in 20 µL reverse transcription (RT) buffer containing 0.5 mM dNTP, 2.5 pmol oligo-dT₁₆ primer, 10 mM dithiothreitol, 20 U RNase inhibitor (Takara, Shiga, Japan), and 200 U Moloney-murine leukemia virus (M-MLV) reverse transcriptase (Life Technologies). Following RT, the reaction solution was inactivated by heating at 70°C for 15 min. To obtain the partial nucleotide sequence of H. japonica AQP-h9 cDNA, a set of PCR primers (sense, 5′-GGATCTGCAGCAGTTTTTGG-3′; antisense, 5′-ATAGCTGCTCCAAGCATAGG-3′) was designed based on the cDNA sequence of Xenopus tropicalis (accession number XM_002937673). The conditions of the PCR amplification were denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 60 s. The PCR products were purified after agarose gel electrophoresis and were sequenced. After a central portion of AQP-h9 cDNA was obtained, 3′- and 5′-rapid amplification of cDNA ends (RACE) techniques were applied to determine the full-length sequence of AQP-h9 cDNA, as reported by Akabane et al. (2007). For 3′-RACE, 1 µg total RNA was reverse transcribed using oligo-dT-adaptor primer (5′-(T)₁₉-GACTCGAGTCGACATCGA-3′) and M-MLV reverse transcriptase (Life Technologies) in 20 µL reaction solution. A 1 µL aliquot of the RT product was then amplified by PCR with 3′-RACE primer (5′-TACAGCTCTGTTGCTCATGC-3′) and adaptor primer (5′-GACTCGAGTCGACATCGA-3′), followed by nested PCR using 3′-RACE nested primer (5′- CCAAGAATCTTTACAGCTC-3′) and the adaptor primer. For 5′-RACE, 500 ng total RNA was reverse transcribed using 5′-RACE primer (5′-ATGTCAGAGCACAGTTCAGC-3′). After the first strand of cDNA was purified with a QIAquick PCR purification kit (QIAGEN, Hilden, Germany), a poly(A) tail was added to the 3′-terminus of the cDNA with terminal deoxynucleotidyl transferase (Toyobo, Osaka, Japan). A 5 µL aliquot of poly(A)-tailed cDNA was then amplified by PCR with the set of 5′-RACE and adaptor primers, followed by nested amplification using 5′-RACE nested primer (5′-AACAGCAACTGGCTCTAGTC-3′) and the adaptor primer. The conditions of the PCR amplification for 3′- and 5′-RACE were denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 90 s. The amplified products were purified and sequenced as described above.

Molecular phylogenetic analysis

The amino acid sequences of aquaglyceroporins from fish to human were aligned using Clustal W (Thompson et al., 1994). An optimal unrooted tree was inferred by the neighbor-joining method (Saitou and Nei, 1987) in the MEGA program ver. 5.0 (Tamura et al., 2011). The evolutionary distances were computed using the p-distance method (Nei and Kumar, 2000), and confidence in the neighbor-joining tree was assessed with 10,000 bootstrap replications (Felsenstein, 1985).

RT-PCR and quantitative real-time RT-PCR (qPCR) for AQP-h9 mRNA

Total RNA was extracted from H. japonica organs, i.e., the brain, heart, lung, stomach, intestine, liver, urinary bladder, kidney, fat body, skeletal muscle, and pelvic skin, and was reverse transcribed with the oligo-dT primer and M-MLV reverse transcriptase (Life Technologies), as described above. Specific primers for H. japonica AQP-h9 (sense, 5′-ATCCAGCAGTTTCGTTTGCG-3′; antisense, 5′-CAATGTTGACAGATACGGAGATGG-3′) and β-actin (AB092519) (sense, 5′-TGGCATCACACCTTCTACAATGAG-3′; antisense, 5′-TCACCAGAGTCCATCACGATACC-3′) were synthesized and used in PCR to amplify 227- and 215-bp products, respectively. RT-PCR was performed using TaKaRa Ex Taq (Takara) according to the manufacturer's instructions. The conditions of the PCR amplification were denaturation at 94°C for 1 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 65°C for 30 s, and extension at 72°C for 30 s. The amplified products were visualized on a 2% agarose gel containing ethidium bromide. Authenticity of PCR products was confirmed by sequencing. The qPCR assay was performed on LightCycler 480 (Roche Diagnostics, Tokyo, Japan) with 10 µL buffer containing 4 µL diluted cDNA, 0.5 µL of each of the primers (final concentration of 0.4 µm), 5 µL FastStart Essential DNA Green Master (Roche Diagnostics), and water in each well of a 384-well plate. The PCR program consisted of denaturation at 95°C for 5 min, and 45 cycles of denaturation at 95°C for 10 s, annealing at 65°C for 10 s, and extension at 72°C for 12 s. Following the cycle amplification, melting curve analysis was conducted from 65°C to 97°C to assess the specificity of PCR amplification. The qPCR assay was carried out in duplicate. The expression level of AQP-h9 mRNA was calculated according to a standard curve and normalized to β-actin mRNA using the LightCycler 480 multiple plate analysis software (Roche Diagnostics). Total RNA without RT was used as a negative control to check for contamination of genomic DNA, and water was used to check primer-dimer formation and reagent contamination.

Production of anti-AQP-h9 serum

A peptide corresponding to the amino acid residues 260–275 of AQP-h9 (ST-225; CIDIHDKKEPDHEMDP, Fig. 1) was synthesized as an antigen (Tanpaku Seisei Co., Gunma, Japan). Anti-AQP-h9 antibody was raised in a rabbit immunized with the ST-225 peptide coupled to keyhole limpet hemocyanin (Pierce Biotechnology, Rockford, IL, USA), according to Tanaka and Kurosumi (1992).

Osmotic water and glycerol permeability of AQP-h9

A cDNA encoding the full-length AQP-h9 was produced by RT-PCR using a sense primer, consisting of a Kozak sequence and AQP-h9-specific sequence (5′-GCCACCATGAGGGAAAGGAGAAGCTG-3′), and antisense AQP-h9 primer (5′-(T)₂₅-ACAGTTCAGCAACGA-3′). The product of the PCR amplification was cloned into the pGEM-3Z vector (Promega, Madison, WI, USA). The pGEM-3Z vector containing the entire open reading frame (ORF) of AQP-h9 cDNA was linearized with EcoR I (Takara), and capped AQP-h9 cRNA was prepared with SP6 RNA polymerase (mCAP mRNA capping kit; Stratagene, La Jolla, CA, USA). Xenopus laevis oocytes at stages V–VI were defolliculated for 1 h with 1 mg/mL collagenase B (Roche Diagnostics) in sterile OR2 solution (100 mM NaCl, 2 mM KCl, 2 mM MgCl₂, and 5 mM Tris-HCl, pH 7.5). Isolated oocytes were microinjected with either cRNA (50 ng) in 50 nL DW or 50 nL DW and were incubated at 18°C for three days in Barth's buffer [88 mM NaCl, 10 mM KCl, 2.4 mM NaHCO₃, 1.7 mM MgSO₄, 0.48 mM Ca(NO₃)2, 0.41 mM CaCl₂, and 7.6 mM Tris-HCl, pH 7.6, 200 mOsm] containing 10 µg/mL penicillin and 10 µg/mL streptomycin, after which they were transferred from 200 mOsm to 70 mOsm Barth's buffer (Saitoh et al., 2014; Shibata et al., 2014a, b) or to isotonic glycerol solution [130 mM glycerol, 23 mM NaCl, 10 mM KCl, 2.4 mM NaHCO₃, 1.7 mM MgSO₄, 0.48 mM Ca(NO₃)₂, 0.41 mM CaCl₂, and 7.6 mM Tris-HCl, pH 7.6, 200 mOsm] (Hansen et al., 2002). The osmotically-elicited increase in volume was monitored at 24°C under a BX50 microscope (Olympus, Tokyo, Japan) with a 4× objective lens and a charge-coupled device camera connected to a computer. The coefficient of osmotic water permeability (Pf) was calculated as reported previously (Zhang et al., 1990; Fushimi et al., 1993). Glycerol permeability (Ps) was estimated according to Carbrey et al. (2003). Some of the AQP-h9 cRNA-injected oocytes were incubated with 0.3 mM HgCl₂ for 10 min before transfer to 70 mOsm Barth's buffer. Expression of AQP-h9 protein in the cRNA-injected oocytes was confirmed by Western blotting and immunohistochemistry.

Fig. 1.

Nucleotide sequence of Hyla japonica AQP-h9 cDNA. The predicted amino acid sequence of the AQP-h9 protein is shown below the nucleotide sequence (accession number LC008216). Six putative transmembrane domains are underlined (TMHMM Server v. 2.0,  http://www.cbs.dtu.dk/services/TMHMM/; Krogh et al., 2001), and two canonical Asn-Pro-Ala (NPA) motifs are outlined. The circle and triangle indicate a putative N-glycosylation site and a potential phosphorylation site for protein kinase C, respectively. The broken line indicates the positions of the specific AQP-h9 primers utilized for RT-PCR and qPCR. The shadowed region corresponds to the amino acid sequence of the AQP-h9 antigen peptide, ST-225. The asterisk indicates the termination codon. The AATAAA polyadenylation signal is double underlined.

Western blot analysis

The AQP-h9 cRNA- or DW-injected X. laevis oocytes were homogenized in a cell lysis buffer [50 mM Tris-HCl, 0.15 M NaCl, 1% (v/v) Triton X-100, 0.1 mg/mL phenylmethylsulfonyl fluoride, and 1 µg/mL aprotinin, pH 8.0] and were centrifuged at 16,000 × g for 10 min to remove insoluble materials. The protein was denatured at 37°C for 60 min in a denaturation buffer [3% (w/v) sodium dodecyl sulfate, 70 mM Tris-HCl, 11.2% (v/v) glycerol, 5% (v/v) 2-mercaptoethanol, and 0.01% (v/v) bromophenol blue, pH 6.8], subjected to electrophoresis on a 12% (w/v) polyacrylamide gel, and then transferred to an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membrane was blocked in 2% (w/v) skim milk (Wako Pure Chemicals, Osaka, Japan). The proteins on the membrane were reacted with rabbit anti-AQP-h9 serum diluted at 1:2,000, followed by incubation with peroxidase-labeled donkey anti-rabbit IgG (Dako, Copenhagen, Denmark). Immunopositive bands were visualized using enhanced chemiluminescence Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK). To check the specificity of the immunoreaction, an absorption test was performed using the antiserum preincubated with an excess amount (10 µg/mL) of the antigen peptide.

Freezing experiment

H. japonica, hibernating at 2–10°C, were transferred into 50 mL polypropylene tubes filled with moistened moss. The tubes were submerged in a cooling bath (CP-300R, TAITEC, Saitama, Japan) at -4°C for 6 h. Following the freezing, some frogs were sampled as a frozen group. The remaining frogs were thawed at room temperature (approximately 23°C) for 1 h after the 6 h freezing, followed by sampling. More than 80% of the frogs survived the freezing experiment. The active frogs sampled in April and the hibernating frogs were used as comparative controls. The frogs of each group were dissected, and then the blood was collected from the heart using capillary tubes (Terumo, Tokyo, Japan). The liver and thigh muscle were excised, immediately frozen in liquid nitrogen, and stored at -80°C until measurement of glycerol content or RT-PCR analysis was conducted. The excised tissues were also fixed in a periodatelysine-paraformaldehyde fixative (PLP: 2% paraformaldehyde, 75 mM L-lysine hydrochloride, and 10 mM sodium periodate in 0.1 M phosphate buffer, pH 7.4) at 4°C for 16 h and were used for immunohistochemical analysis.

Measurement of glycerol concentrations in the serum and tissue extract

The blood samples of the frogs were centrifuged at 450 × g at 4°C for 10 min. The sera were then collected and stored at -20°C until further use. Each tissue sample from the liver and muscle was homogenized in 400 µL of ice-cold 0.7 × phosphate buffered saline (PBS; 0.01 M sodium phosphate buffer and 0.14 M NaCl, pH 7.5). After centrifugation at 20,000 × g at 4°C for 10 min, the supernatant was collected and used to measure the glycerol content. Glycerol concentrations in the serum and tissue extract were determined with a free glycerol reagent (Sigma-Aldrich, St. Louis, MO, USA). After incubation of the reaction solution containing 80 µL reagent and 10 µL serum, tissue extract, or glycerol standard at 37°C for 30 min, absorbance at 562 nm was measured with a microplate reader (Molecular Devices, Sunnyvale, CA, USA). The glycerol concentration was calculated from a standard curve. The tissue content of glycerol was standardized by the protein amount of each tissue, which was determined with a Pierce BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions.

Fig. 2.

Comparison between amino acid sequences of AQPs from Hyla japonica (A) and neighborjoining unrooted tree of aquaglyceroporin proteins from fish to human (B). (A) A pair of Asn-Pro-Ala (NPA) motifs (diamonds) are conserved in H. japonica AQPs; AQP-h9 (LC008216), AQP-h3BL (AQP3, BAF63030), AQP-h1 (AQP1, BAC07470), AQP-h2K (AQP2, BAF80993), AQP-h2 (AQPa2U, BAC82379; Shibata et al., 2014a), and AQP-h3 (AQPa2S, BAC07471; Shibata et al., 2014a). Six membrane-spanning domains are predicted in AQP-h9 (top lines), as in other AQPs. The circle, open triangle, and solid triangle indicate N-glycosylation sites, phosphorylation sites for protein kinase C, and phosphorylation sites for protein kinase A, respectively. Gaps marked by hyphens are inserted to optimize homology. (B) Aquaglyceroporins are divided into AQP3, AQP7, AQP9, and AQP10, and AQP-h9 is assigned into AQP9. Anuran-specific AQPxlo (Virkki et al., 2002) is also classified to AQP9, together with AQPxto (ENSXETG00000016307). The length of each branch is proportional to the mean number of differences per residue, and the numbers around the interior branches are bootstrap probabilities (percent; 10,000 replicates). The organism and accession number are indicated for each AQP.

Histology and immunofluorescence staining

The fixed liver and muscle were dehydrated through a graded ethanol series and were embedded in Paraplast plus (McCormick Scientific, St. Louis, MO, USA). Sections were prepared according to Nakakura et al. (2009). The sections were stained with Mayer's hematoxylin and eosin, dehydrated with ethanol, and then mounted in Entellan (Merck, Darmstadt, Germany). For immunofluorescence staining, the sections were covered with rabbit anti-AQP-h9 (1:1000) or rabbit anti-AQP-h3BL (1:2000; Akabane et al., 2007) for 16 h, followed by incubation with indocarbocyanine (Cy3)-labeled donkey anti-rabbit IgG (1:400; Jackson Immunoresearch, West Grove, PA, USA) for 2 h. For counterstaining of the nucleus, 4′, 6-diamidino-2-phenylindole (DAPI) was added to the secondary antibody solution. The specimens were washed in PBS and were mounted in PermaFluor (Thermo Fisher Scientific). The preparations were observed under an Olympus BX61 microscope equipped with a BX-epifluorescence attachment (Olympus). The specificity of immunostaining was checked using the antiserum that had been preincubated with 10 µg/mL of the antigen peptide.

Statistical analysis

Values were expressed as means ± SEM. The data were compared using Tukey's test. Statistical significance was set at P < 0.05.

Cloning of AQP-H9 cDNA

The nucleotide sequence and deduced amino acid sequence of H. japonica AQP-h9 cDNA are shown in Fig. 1. The cDNA consisted of 885 bp of a coding region, 63 bp of a 5′-untranslated region (UTR), and 780 bp of a 3′-UTR followed by a poly(A) tail. The ORF encoded a protein of 294 amino acid residues with the molecular mass calculated to be 31.5 kDa. As in other AQPs, certain features were conserved in AQP-h9; these were six transmembrane domains and a pair of canonical Asn-Pro-Ala (NPA) motifs at residues 79–81 and 211–213 (Figs. 1 and 2A). A putative N-linked glycosylation site and a potential phosphorylation site for protein kinase C were noted at Asn-137 and at Thr-287, respectively. AQP-h9 showed 20%, 21%, 44%, 19%, and 20% amino acid identity with AQP-h1 (Tanii et al., 2002), AQP-h2K (Ogushi et al., 2007), AQP-h3BL (Akabane et al., 2007), AQP-h2 (Hasegawa et al., 2003), and AQP-h3 (Tanii et al., 2002), respectively (Fig. 2A). On the other hand, AQP-h9 exhibited 59%, 56%, 55%, and 52% amino acid identity with X. tropicalis AQP9 (ENSXETG00000010861), chicken AQP9 (Sugiura et al., 2008), human AQP9 (Ishibashi et al., 1998), and medaka AQP9 (ENSORLG00000008259), respectively. Molecular phylogenetic analysis indicated that AQP-h9 forms the AQP9 cluster together with the AQP9 proteins of other vertebrates (Fig. 2B).

Expression of AQP-H9 mRNA

To investigate the distribution of AQP-h9 mRNA expression, RT-PCR was performed using specific primers. AQP-h9 mRNA expression was observed in all the organs tested, such as the brain, heart, lung, stomach, intestine, liver, urinary bladder, kidney, fat body, skeletal muscle, and pelvic skin (Fig. 3). Among these organs, AQP-h9 mRNA was expressed at relatively high levels in the liver, stomach, and pelvic skin.

Fig. 3.

AQP-h9 mRNA expression in various organs of Hyla japonica. RT-PCR products were separated on a 2% agarose gel and were stained with ethidium bromide. An amplified band for AQP-h9 mRNA was detected in all the tissues examined, including the liver and thigh muscle, β-actin mRNA was used as an internal control.

Water and glycerol permeation through AQP-h9

Transmembrane water flow through the AQP-h9 protein was evaluated by a swelling assay using X. laevis oocytes. Microinjected oocytes were incubated at 18°C for three days and were then transferred from isotonic (200 mOsm) to hypoosmotic (70 mOsm) Barth's buffer. The relative volume of the AQP-h9 cRNA-injected oocytes was markedly increased by hypotonic stimulation (Fig. 4A). The Pf value of the AQP-h9 cRNA-injected oocytes was approximately threefold higher than those that were injected with DW (Fig. 4B). The osmotic water permeability was inhibited by adding 0.3 mM HgCl₂ to incubation buffer (Fig. 4A, B). Effect of AQP-h9 on the glycerol permeability was analyzed by a swelling assay with isotonic (200 mOsm) glycerol solution (Fig. 4C, D). In this experimental system, glycerol enters the oocyte along the concentration gradient with a secondary influx of water, leading to oocyte swelling. Glycerol permeability in the AQP-h9 cRNA-injected oocytes was approximately fourfold higher than those injected with DW (Fig. 4D). Inhibition of the glycerol permeability was confirmed by adding 0.3 mM HgCl₂ to incubation buffer (Fig. 4C, D).

To evaluate whether the AQP-h9 protein was expressed in the cRNA-injected oocytes, sections of the AQP-h9 cRNA-injected oocytes were immunostained with anti-AQP-h9 (Fig. 4E). Immunopositive labeling was observed in the plasma membrane (Fig. 4E, a and b), agreeing well with the result of the swelling assay. The immunoreactivity was completely abolished by preabsorption of the antiserum with an excess amount of the immunogen peptide (Fig. 4E, c). No labeling was detected in the DW-injected oocytes (Fig. 4E, d). These results verified the specificity of the antibody. The expression of AQP-h9 protein in the oocytes was also examined by Western blot analysis. As shown in Fig. 4F, a band was detected at 31 kDa in an extract of AQP-h9 cRNA-injected oocytes (Fig. 4F, lane 1). The molecular mass of this protein was consistent with that predicted from the deduced amino acid sequence of AQP-h9. This band was not found in the DW-injected oocytes (Fig. 4F, lane 2) and vanished when the antiserum was preabsorbed with the antigen peptide (Fig. 4F, lane 3). These results confirm the specificity of the antibody and suggest that the band of 31 kDa represents AQP-h9.

Glycerol concentrations in the blood and tissues

The effects of freezing and/or thawing on the physiological condition of H. japonica were assessed by measuring the levels of glycerol in the serum, liver, and skeletal muscle. Plasma glycerol concentration did not change before and after hibernation, but it increased approximately 10-fold in the thawed frogs (Fig. 5A). In the frozen group, the blood remained in the liquid phase, but the heartbeat had already stopped. Therefore, it was impossible to collect a sufficient amount of blood to measure glycerol concentrations. As for the tissues of the hibernating frogs, the glycerol level was markedly higher in the liver than in the muscle (Fig. 5B). No difference was detected in the hepatic glycerol concentrations among the hibernating, frozen, and thawed groups. By contrast, for the muscle, the glycerol concentration increased nearly 10-fold in the frozen frogs and about 50-fold in the thawed frogs as compared with the hibernating frogs (Fig. 5B).

Fig. 4.

Water and glycerol permeability analysis of AQP-h9 using Xenopus laevis oocytes. (A) Time course of the osmotic swelling by hypotonic stimulation. Oocytes were microinjected with DW or AQP-h9 cRNA. Some of the AQP-injected oocytes were incubated with 0.3 mM HgCl₂. (B) Osmotic water permeability (Pf) was calculated from the initial rate of oocyte swelling. Data are shown as the mean ± SEM (n = 6). Values with the different letters are significantly distinguishable from each other (Tukey's test, P < 0.05). (C) Time course of swelling in isotonic glycerol solution. Oocytes were microinjected with DW or AQP-h9 cRNA. Some of the AQP-h9-injected oocytes were incubated with 0.3 mM HgCl₂ (D) Glycerol permeability (Ps) was calculated from the initial rate of oocyte swelling. Data are shown as the mean ± SEM (n = 6). Values with the different letters are significantly distinguishable from each other (Tukey's test, P < 0.05). (E) Immunofluorescence images of AQP-h9 protein in the DW- or cRNA-microinjected oocytes. AQP-h9 immunoreactivity was intensively detected along the plasma membrane of the cRNA-injected oocytes (a); the Nomarski differential interference image corresponding to Fig. 4E, a (b); in the absorption test, immunoreactivity was abolished to background levels (c); and no immunopositive labels were observed in the DW-injected oocytes (d). Arrows indicate the plasma membrane. Scale bar = 100 µm. (F) Western blot analysis of the AQP-h9 cRNA- (lanes 1 and 3) or DW- (lanes 2 and 4) injected oocytes using anti-AQP-h9 serum (lanes 1 and 2) or anti-AQP-h9 serum preabsorbed with the antigen peptide (lanes 3 and 4). An arrow indicates an AQP-h9-specific band. Asterisks indicate non-specific signals.

Fig. 5.

Effects of freezing on glycerol concentrations in the serum (A) and tissue extracts (B) of Hyla japonica. (A) Serum glycerol concentration significantly increased when hibernating frogs were frozen and thawed. (B) Hepatic glycerol concentration did not change among the hibernating, frozen, and thawed frogs, whereas glycerol concentration of the thigh muscle significantly increased in the thawed frogs compared with the hibernating frogs. Data represent means ± SEM (n = 7). In each organ, values with the different letters are significantly distinguishable from each other (Tukey's test, P < 0.05).

Histology and immunohistochemistry for AQP-H9

Cytological features of the liver of H. japonica were first examined by traditional histochemical methods using hematoxylin and eosin. The parenchyma consisted of irregular cords or tubes of hepatocytes, forming internal intercellular bile canaliculi. Erythrocytes were observed in adjacent sinusoids and veins (Fig. 6A–D). After hibernation, the eosinophilic substance was lost, and the cytoplasm of hepatocytes was not densely stained except for hematoxyphilic materials near the bile canaliculi (Fig. 6B–D). Additionally, the erythrocytes aggregated in a sinusoidal net and veins (Fig. 6B). The erythrocyte clusters expanded in the frozen group (Fig. 6C) but were not clearly recognized in the thawed group (Fig. 6D). For the skeletal muscle, myocyte atrophy was observed after hibernation (Fig. 6F) and was more evident in the frozen group (Fig. 6G).

Fig. 6.

Hematoxylin and eosin-stained images of the liver (A–D) and thigh muscle (E–H) in the active (A, E), hibernating (B, F), frozen (C, G), and thawed (D, H) Hyla japonica. In the parenchyma of the liver, hepatocytes were arranged as irregular cords or tubes (A–D). The cytoplasm of hepatocytes was densely stained in the active group (A), but not in the other groups (B–D). The erythrocyte clusters (arrowheads) were evident in the sinusoids of the hibernating group (B) and frozen group (C). For the skeletal muscle, the cytoplasm of myocytes was stained in the active group (E), but the staining was weaker in the other groups (F–H). Myocyte atrophy was Induced in the hibernating group (F) and progressed in the frozen group (G). Scale bar (A–D) = 50 µm; (E–F) = 20 µm.

Immunofluorescence staining was then performed to examine the expression and localization of AQP-h9 in the liver and skeletal muscle. AQP-h9-immunoreactive signals were scarcely recognized in the liver of active frogs (Fig. 7A). Labels for AQP-h9 were detectable in erythrocytes in the liver of the hibernating frogs (Fig. 7C). Labels became more intense and located over the erythrocytes after freezing (Fig. 7E) but nearly disappeared in the thawed frogs (Fig. 7G). For the skeletal muscle, immunopositive labels for AQP-h9 were hardly detected in the active frogs (Fig. 8A), while weak labels were observed within myocytes in the hibernating frogs (Fig. 8C). AQP-h9 labeling was markedly enhanced in the frozen frogs (Fig. 8E) and was decreased in the thawed frogs (Fig. 8G). Immunoreactivity for AQP-h9 disappeared in the liver and muscle when the antiserum was preabsorbed with the antigen peptide (data not shown), confirming the specificity of the reaction. On the other hand, immunopositive AQP-h3BL was nearly undetectable in the liver or muscle in all the frog groups (data not shown).

Expression of AQP-H9 mRNA

With regard to the liver and thigh muscle, the mRNA expression of AQP-h9 was analyzed by RT-PCR and qPCR. AQP-h9 mRNA was intensely expressed in the liver and weakly expressed in the muscle (Fig. 9A). The expression levels of AQP-h9 mRNA in the liver and muscle were not changed by freezing or thawing (Fig. 9A and B).