10.1007/s11434-011-4911-3 cDNA cloning and expression of erythropoietin in the plateau zokor ( Myospalax baileyi ) from the Qinghai-Tibet

2011 All organisms respond to variation in their environments and manage environmental stress through metabolic adjustments. The plateau zokor ( Myospalax baileyi ) is an endemic and keystone subterranean rodent species that inhabits the Qinghai-Tibet Plateau between 2800 and 4200 m above sea level. It is a hypoxic-tolerant mammal with a high ratio of oxygen utilization that enables it to cope with its harsh surroundings. To explore the molecular mechanism of altitude acclimatization of the plateau zokor, we cloned the zokor erythropoietin ( Epo ) gene and used real-time PCR to compare Epo mRNA levels in zokors inhabiting 16 different altitudes. The full-length zokor Epo open reading frame was 579 bp that encoded a precursor peptide of 192 amino acids with a signal peptide of 26 residues. The Epo gene of the plateau zokor was 81%–95% homologous to that of human, mouse, rat, root vole and the Golan Heights blind mole rat, with the highest homology (95%) to species of the genus Spalax. Epo mRNA was detected mainly in the zokor kidney and spleen among 8 selected tissues. The level of Epo mRNA increased in the liver and kidney with increases in altitude. The increase in the kidney was 5 times that in the liver. Remarkably, expression of Epo mRNA in the kidney of zokors living at the highest altitude (4268 m) was 12-fold higher than that of zokors living at the lowest (2492 m) altitude. These findings provide essential information for understanding the possible role of Epo in adaptation to hypoxia in the plateau

All organisms have to deal with environmental stress through metabolic adjustment and ultimately they have to adapt to their environment [1]. The Qinghai-Tibet Plateau has an average altitude of 3000 m, making it the highest plateau on earth [2]. Strong ultraviolet radiation, hypoxia and cold are typical of plateaus and have a profound effect on animal survival. Over evolutionary time many animals have developed unique mechanisms in response to these harsh environmental conditions to ensure optimal growth and reproduction [3].
The plateau zokor (Myospalax baileyi) is an important subterranean rodent endemic to the Qinghai-Tibet Plateau. It is tolerant of hypoxia and shows a remarkably high oxygen utilization ratio to cope with the plateau environment, making it a good model for research into adaptation to hypoxia [4]. In their sealed hypoxic-hypercapnic burrow, Zo-kors spend their entire life underground, exposed to fluctuating O 2 and CO 2 levels [5,6]. They are active throughout the year, excavating burrow systems and storing food. As is common in subterranean rodents, the plateau zokor exhibits morphological, physiological and behavioral adaptations to digging and living underground [7]. For example, the zokor has evolved physiological changes to its respiratory and cardiovascular systems to cope with hypoxia [8][9][10]. The zokor is dramatically superior to Rattus species in microvessel density of cardiac muscle and myocardial performance, and harbors a much higher capillary and mitochondrial density [8]. The zokor has a higher erythrocyte count, an increased lung diffusion capacity, and utilizes a myoglobin that is different in structure to that of Rattus [10]. Moreover, the number of red blood cells, the density of hemoglobin in the red blood cells and the myoglobin content in heart and skeletal muscle are much higher than in other high-altitude mammals inhabiting a plateau environment [11].
Erythropoietin (Epo) is a hematopoietic cytokine that regulates red blood cell production [12]. Epo has 166 amino acid residues and a molecular weight of 18.4 kD; however, the overall molecular weight of Epo is 30.4 kD because of three N-linked carbohydrate chains. Two internal disulphide bonds between cysteine residues 7-161 and 29-33 are known to be necessary for biological activity [13]. Epo is expressed mainly in fetal liver and adult kidney [14,15]. The regulation of Epo gene expression occurs mainly at the transcriptional level by DNA-dependent mRNA synthesis [16][17][18] and is controlled by an oxygen detection system that responds to changes in venous rather than arterial partial pressure of oxygen (PO 2 ) [19,20]. Clearance of Epo appears to be possible via three routes: excretion through the kidney [21], metabolism by the liver [22] or consumption by the erythron [23]. Epo is essential for the development of normal erythropoiesis, and underproduction of Epo results in anemia [16]. Regulation of erythropoiesis and red blood cell mass relies on modulating Epo gene expression in response to tissue oxygen tension. Developmental, tissue-specific, and environmental signals all contribute to the precise regulation of the Epo gene. Research on ontogenetic expression of the Epo gene and of hypoxic stress tolerance in the subterranean blind mole rat of the Spalaxeh renbergi superspecies has shown that higher Epo mRNA levels exist in the Spalax fetal liver and kidney compared with Rattus. In addition, adult Rattus kidney and liver, and adult Spalax liver express similar levels of Epo mRNA under normoxia and hypoxia [24]. In normoxia, both Spalax and Rattus kidneys produce small amounts of Epo. Notably, under hypoxic conditions, Epo expression in the adult Spalax hypoxic kidney, the major site of erythropoietin production in adult mammals, was remarkably higher than in that of Rattus. Therefore, it can be assumed that Epo is a key factor in coping with underground hypoxic stress and that Epo contributes to the adaptive strategy of hypoxia tolerance [25].
Epo has been cloned from many mammals, including human, monkey, rat, sheep, dog and pig and it shows a high degree of homology among mammals [18,[25][26][27]. However, there have been few reports concerning the Epo gene from species living in high altitude, hypoxic environments. The plateau zokor is a hypoxic-tolerant mammal with a high ratio of oxygen utilization. In this study, the Epo gene of the plateau zokor was cloned, and the predicted amino acid sequences were compared with those of other animals. Further, using real-time PCR, we investigated expression levels of Epo mRNA from 8 tissues and across 16 different altitudes to explore the significance of Epo in the adaptation of the plateau zokor to the harsh environment of the Qinghai-Tibet Plateau.

Experimental sites and animal sampling
All procedures involved in the trapping and handling of animals were approved by the Chinese Zoological Society and were conducted in accordance with the Chinese Practice for the Care and Use of Laboratory Animals. Eighty-eight adult zokors (58 females and 30 males) were captured from 16 locations in the eastern region of the Qinghai-Tibet Plateau over two field excursions from April to June in 2006 and 2007. We collected animals from sites dominated by alpine meadows, alpine shrub and farmland where the main differences were altitude (ranging from 2492 to 4268 m) and ambient temperature (ranging from −1.3 to 11.8°C). At each location we measured elevation, latitude and longitude using a handhold GPS (eXplorist210, Magellan Corp, USA) ( Table 1). Live-trapping was conducted between 09:00 and 16:00 h from April to June. Captured animals were firstly recorded for body weight (g) and body length (mm), then anesthetized using chloral hydrate (5%) and killed by cervical dislocation. Dissections were conducted at the trap site. Eight tissue samples (brain, heart, lung, liver, spleen, kidney, muscle and white adipose tissue) were immediately frozen in liquid nitrogen.

Preparation of total RNA and cDNA synthesis
Total RNA was extracted from a 100-mg portion of frozen tissue using TRIZOL reagent (Invitrogen, Carlsbad, USA), and then diluted in 100 μL RNase-free water and analyzed by agarose gel electrophoresis. Sharp bands of 18S and 28S rRNA were used as indicators of high quality preparations. RNA concentration was determined by spectrophotometry using a NanoDrop ND-1000 Spectrophotometer (Nano-Drop Technologies, Rockland, USA). RNA samples were treated with RNase-free DNase I (TaKaRa, Dalian, China), then stored at −80°C.
Five micrograms of total RNA were used for first-strand cDNA synthesis using a SuperScript™ II RT Kit (Invitrogen) in a 30-μL volume containing 5×first-strand Buffer, Oligo(dT) 18 (20 μg/mL), 10 mmol/L dNTPs, 0.1 mmol/L DTT, RNaseOUT (40 units/μL) and reverse transcriptase (40 units/μL). The reaction was carried out at 65°C for 5 min, then at 42°C for 50 min. The reverse transcriptase was then inactivated at 70°C for 15 min. Aliquots of undiluted cDNA were used for PCR and real-time PCR.

Cloning the plateau zokor erythropoietin gene
To obtain a complete open reading frame (ORF) of the plateau zokor Epo gene, forward (Epof 20-39: GAGATG-GGGGTGCCCGAACG, accession no. NM_017001) and reverse (Epor 583-602: GTCACCTGTCCCCTCTCCTG) primers were designed with Primer Premier (version 5.0, Premier Biosoft International, Palo Alto, USA) according to the alignment of highly conserved coding sequence regions of the Epo gene among humans (accession no. NM_ 000799), mice (accession no. NM_007942), rats (accession no. NM_017001) and Spalax sp. (accession nos. AJ715792, AJ715793, AJ715794, AJ715795). Total kidney RNA was isolated from 6 zokors, and Epo cDNA was obtained by RT-PCR. The Epo gene product was amplified by PCR using LA Taq DNA polymerase (TaKaRa) in a mixture of 200 μmol/L dNTP, 0.3 μmol/L primers (Beijing Genomics Institute, Beijing, China) and 2×GC Buffer I (Mg 2+ plus) with 5 μg undiluted cDNA template. The PCR reactions were started with 10 min at 95°C followed by 35 cycles consisting of 45 s at 94°C, 30 s at 55°C, and 60 s at 72°C. The PCR product of expected size was purified using Axyprep DNA Gel Extraction Kit (Axygen Biosciences, California, USA) and ligated into pMD19-T (TaKaRa). Five clones from each animal were then sequenced and the full-length Epo cDNA was determined by alignment with the above species. The entire sequence was submitted to the GenBank database (accession no. EU183314).

Sequence analysis
Translation of cDNA nucleotide sequence was performed using the EditSeq program of DNASTAR (DNASTAR, Madison, USA). The nucleotide and deduced amino sequences were compared with sequences in the GenBank database using the BLAST program available from the National Center for Biotechnology Information (NCBI, US National Institute of Health). The signal peptide in the deduced amino acid sequence was predicted with SignalP available from Centre for Biological Sequence Analysis, Technical University of Denmark. Multiple alignments were performed using CLUSTALX (version 1.81, Plate-Forme de Bio-Informatique, Illkirch, France). Functional motifs were predicted in the deduced amino acid sequence using MotifScan in the PROSITE database of protein families and domains (available from http://myhits.isb-sib.ch/ cgi-bin/motif_scan).

Primers and real-time PCR
The endogenous β-actin gene was used as the reference gene. Real-time PCR primers for the Epo and β-actin genes were designed using Primer Express Software (v3.0, Applied Biosystems, Carlsbad, USA). For the Epo gene, forward (F: 5′-ACAATGGAGGTGGAAGAACAGG-3′) and reverse (R: 5′-ATGGTTGGGAGGAATTGGCT-3′) primers amplified a 106-bp fragment. For the β-actin gene, forward (F: 5′-CTAAGGCCAACCGTGAAAAGAT-3′) and reverse (R: 5′-GACCAGAGGCATACAGGGACA-3′) primers amplified a 105-bp fragment. Real-time PCR was carried out in 30 μL, containing 5×Real Time PCR buffer, 250 mmol/L Mg 2+ , 10 mmol/L dNTPs, 10 pmol of each primer, 1:20 SYBR GreenI, 1 U Ex Taq (TaKaRa) and 2 μL of cDNA or 10 fold-diluted plasmids on an ABI 7300 instrument (Applied Biosystems). Each sample was tested in triplicate, using the following conditions: initial denaturation for 10 min at 95°C followed by 40 cycles of 20 s at 95°C, 30 s at 60°C and 30 s at 72°C. The fluorescence signals were read and collected at the end of each extension step at 72°C. After amplification, a melting curve analysis with a temperature gradient of 15 s at 95°C, 30 s at 60°C and 15 s at 95°C was performed to confirm that only the specific products were amplified.

Efficiencies of real-time RT-PCR
The quantities of zokor Epo mRNA were normalized with β-actin mRNA to compensate for variations in input RNA amounts. To obtain reliable results for the comparative C T method, efficiencies of amplification of housekeeping and target genes have to be similar. The efficiency has been calculated for each PCR by the following equation, where S is the slope of the standard curve: Efficiency (E) = [10 (1/Slope) ] -1. (1) By analysis of serial 10-fold diluted plasmids, standard curve equations and linear regression coefficients were calculated automatically by the Real-Time PCR Detection SDS System. Here, efficiency (E) was typically >0.90 (Slope = 3.31 ± 0.20, R 2 =0.94 ± 0.02), indicating that PCR amplification of Epo and β-actin were approximately equal and that the ∆∆C T method was appropriate.

Data analysis
Using the 2 −∆∆C T method [28], normalization was achieved by dividing the average value of Epo by the average value of β-actin in each sample. To compare levels between tissues, we used brain as the calibrator sample. Similarly, the expression of Epo in the liver and kidney at different altitudes was compared with the average ∆C T value for the altitude 2492 m. 2 −∆∆C T values larger than 1 indicated a higher expression level.
Data were analyzed with the program SPSS (version 13.0, SPSS, Chicago, USA). One-way ANOVA was used to test the difference among the 8 tissues. Post Hoc Tests (LSD) were used to compare the mean differences between tissues. Forwards stepwise multiple linear regression analysis was used to determine the effect of altitude, body mass, body length, mean annual temperature and mean annual rainfall on zokor Epo mRNA levels in the kidney and liver. Differences were considered to be significant for a P < 0.05.

Cloning and sequence analysis of the zokor Epo gene
The core region of the zokor Epo gene was cloned and sequenced. The full-length coding sequence was 579 bp with the ATG start codon located at position 7 and the TGA stop codon at position 578. The sequence was submitted to the GenBank database under accession no. EU183314.
Predicted motifs of zokor Epo included three N-glycosylation sites, two casein kinase II phosphorylation sites, two protein kinase C phosphorylation sites, one erythropoietin/thrombopoeitin signature site and one cell attachment sequence site. Two single nucleotide polymorphisms sites (SNPs, defined as a single base-pair difference between two copies of a DNA sequence from two individuals) were identified at 36 M-A/G and 168 M-C/A.

Epo expression in various tissues
The relative expression of Epo (2 −∆∆C T ) in selected tissues of the adult plateau zokor varied significantly (F 7,88 = 51.719, P < 0.001, Figure 3). There was a high level of Epo mRNA per gram of tissue in the kidney (4.53 ± 1.97) and spleen (3.50 ± 1.30). Brain (1.00 ± 0.00), lung (0.57 ± 0.15) and liver (0.42 ± 0.15) exhibited medium levels, while heart (0.09 ± 0.04), muscle (0.06 ± 0.02) and white adipose tissue (0.05 ± 0.02) showed low levels. The amount of Epo mRNA/g tissue in kidney and spleen was 13-fold and 8-fold higher than in liver, respectively. For each adult individual, whether male or female, the organ coefficient was relatively constant. In selected tissues, the organ coefficient of liver and kidney was much higher than in other tissues. Therefore, considering organ weight, the total amount of Epo mRNA in the liver was half that detected in kidney and 2.3-fold that in spleen. The kidney and liver were the dominant organs of the relative contribution to total body Epo mRNA in the plateau zokor.

Comparison of Epo gene expression in plateau zokors from different altitudes
To reveal the major environmental factors affecting expression of zokor Epo, stepwise multiple regression analysis Figure 2 Multiple alignments of Epo amino acid sequences. Residues identical to Mus musculus Epo are presented as dots (.). The predicted motifs are shown using shadings of different colors (protein kinase C phosphorylation site (PKC), yellow; casein kinase II phosphorylation site (CK2), red; N-glycosylation site, green). Underlined amino acid sequences indicate the motif of the erythropoietin/ thrombopoeitin signature site. Convergent evolution sites between M. baileyi and Spalax sp. are shown using grey shading. The numbers at the right are the total numbers of amino acids.

Figure 3
Epo mRNA levels (2 −∆∆CT ) in different tissues of the plateau zokor. Data are presented as mean ± SEM, n = 12 in each group. Bars with different letters (aa, bb, cc) indicate significant differences (P < 0.01) with three grades. WAT, white adipose tissue. was used to determine how altitude (Al, in m), mean annual temperature (Tm, °C) and mean annual rainfall (Rn, in mm) influenced zokor Epo expression in liver and kidney ( Table  1). The results showed that the levels of Epo mRNA in kidney (r = 0.8373, F 1,86 = 144.00, P < 0.001; Figure 4(a)) and liver (r = 0.7790, F 1,86 = 144.00, P < 0.001; Figure 4(a)) were significantly increased with altitude. The slope for kidney (0.0055) was 5 times higher than that for the liver (0.0011). Remarkably, the level of Epo mRNA in the kidney of zokors living at the highest altitude (4268 m) was 12-fold higher than that of zokors at the lowest altitude (2492 m). Mean annual temperature (Figure 4(b)) and mean annual rainfall (Figure 4(c)) were not significantly correlated with Epo expression in liver and kidney (Table 2, Figure 4).

Figure 4
Relative quantification the relationship of Epo mRNA levels in the kidney and liver of the plateau zokor with respect to different altitudes, mean annual temperature, body length, hind-foot length, body mass and mean annual rainfall using the 2 −∆∆CT method. Furthermore, no correlation was found between Epo expression and body weight (Figure 4(d)), body length ( Figure  4(e)), and hind-foot length (Figure 4(f)).

Discussion
Erythropoietin (Epo) is a glycoprotein hormone essential for normal erythropoiesis. In this study, we cloned the Epo gene of the plateau zokor and compared its expression pattern among 8 tissues and across 16 different altitudes using real-time PCR. The full-length zokor Epo gene is 579 bp that encodes a precursor peptide of 192 amino acids with a signal peptide of 26 residues. The nucleotide and deduced amino acid sequences were highly conserved with those of human, mouse, rat, root vole, and Golan Heights blind mole rat. Of note, zokor Epo is 95% identical to Spalax Epo. Although zokor and Spalax sp. belong to different taxonomic genera, the predicted secondary and tertiary structure of zokor Epo is highly similar to that of the Spalax genus [29]. The similarity in Epo structure of subterranean rodents may be related to their similar hypoxic living environments. In addition, research on hypoxic stress tolerance of Spalax has found higher levels of Epo expression in Spalax kidney under sustainable hypoxia [25]. Similarly, we found that there was a tendency for the levels of Epo mRNA to increase in the high-altitude zokor kidney. As altitude increases, the concentration of oxygen in hypoxic-hypercapnic burrows will decrease. This suggests that the shared characteristics of EPO between the plateau zokor and the blind subterranean mole rat (Spalax) may be a result of genetically evolved adaptation to cope with hypoxic stress of the subterranean lifestyle.
Epo is synthesized mainly in fetal liver [14] and this expression is shifted to the kidney shortly after birth [15]. In the adult mammal, the kidney is the major site of Epo production. Extrarenal sites produce smaller amounts of Epo in the adult, including liver, lung, spleen, brain and testis [30]. Our results showed the same conclusion that Epo was mainly produced by the kidney in adult zokors. The amount of Epo mRNA/g tissue in kidney was 13-fold higher than that in liver. In the spleen, we also detected a considerable amount of Epo mRNA/g tissue with a level 8-fold higher than that in liver, which was different from previous reports of Epo tissue-specific expression in human, mouse, rat and other species. Furthermore, there were medium levels in brain, lung and liver and low levels in heart, muscle and white adipose tissue. In conclusion, the expression of Epo in the adult plateau zokor has tissue specificity. As far as the relative contribution of the different organs to total body EPO production, the organ coefficients in liver and kidney were much higher than in other selected tissues. In our results, the total amount of Epo mRNA in the liver was half that detected in kidney and 2.3-fold that in spleen. Therefore, the kidney and liver were the dominant organs for Epo expression in the adult plateau zokor.
All organisms encounter changes in their environment. Responding to environmental challenges and stresses can result, at least over evolutionary time, in the establishment of new and unique metabolic adaptations [1,31]. Different genotypes will show different adaptations to the ever changing environment in which organisms live. This genome-phenome relationship that results from different ecological-genetic patterns demonstrates that this system is a tightly structured one and dependent on abiotic and environmental diversity [29]. If differences in Epo gene sequence and function between zokor and non-zokor species are a result of adaptation to plateau living, we would expect some fine-scale within-species variation in expression. The cold and hypoxic conditions, characteristics of the Qinghai-Tibet Plateau, provide us with a good opportunity to examine some of the drivers of change of Epo expression. We found that zokors living at higher altitudes show relatively higher levels of Epo expression. The relationship between Epo expression in the kidney and altitude was 5 times stronger than that in the liver. The known correlation between altitude and barometric pressure or inspired oxygen pressure is negative [32,33]. As altitude increases, air oxygen content decreases and hypoxia develops. Therefore, differences in altitude and hypoxia across our 16 sampling sites were the most important factors affecting Epo expression in this species and had a profound effect on the adaptation of the zokor to plateau living.
Plateau zokors mostly spend their life in sealed burrows. In their hypoxic-hypercapnic burrows, the temperature and humidity is relatively constant, but the concentration of oxygen and carbon dioxide fluctuates with the changing seasons, rainfall, and the ambient temperature [34]. The O 2 concentration is probably much lower when the burrows are flooded with rainwater or the earth is frozen in winter. High rainfall will drastically limit gas solubility and permeability [29]. Ambient temperature is related to the freeze-thaw of the seasonal permafrost in the Qinghai-Tibet Plateau. When the surface is frozen or thawing, soil ventilation and gas permeability are restricted, resulting in an increase of hypoxic stress [35]. Moreover, zokors will increase oxygen consumption because of the extreme cold [36]. Consequently, plateau zokors are confronted by persistent hypoxia during winter. Although rainwater and frozen earth limit gas permeability and low temperature increases oxygen consumption, our data showed that neither mean annual temperature nor mean annual rainfall were related to the level of Epo. These two factors may have an indirect influence, rather than by directly producing an effect on the level of gene expression. In addition, there was sexual dimorphism in body weight, body length, relative fatness, tail length and hind feet length. Age and sex are positively associated with body mass and body length in zokors [37], but we did not find correlations between levels of Epo expression and body mass, body length, age or sex.

Conclusions
In conclusion, zokor Epo has a high degree of homology to Spalax Epo. Altitude is a major factor that affects Epo expression in the plateau zokor. These results suggest that Epo may play an essential role in the plateau zokor in ecological adaptation to the harsh plateau environment.