During 40 million years of life underground, Spalax evolved physiological strategies underlying their respiratory and cardiovascular systems to cope with hypoxia (1–3). Spalax is dramatically superior to Rattus in vascular endothelial growth factor (VEGF) activity (4) and myocardial performance (5), and harbors much higher capillary and mitochondrial density than does Rattus (6). It has a higher erythrocyte count (7), increased lung diffusion capacity, and different structure of hemoglobin (8), haptoglobin (9), myoglobin (10), neuroglobin, and cytoglobin. Furthermore, there are differences among the four Israeli Spalax allospecies in their s.c. gas tension (7), hemoglobin, and hematocrit concentrations, with higher values in the northern S. galili compared with the southern S. judaei (7).

Erythropietin and its expression inducer hypoxia-inducible factor-1 (HIF-1). Erythropoietin (Epo) is the main factor involved in erythropoiesis, regulating the level of circulating red blood cells. Epo is expressed mainly in fetus liver and adult kidneys (11, 12) and is regulated by hypoxia stimulus at the RNA level (13). Epo has also angiogenic activity that stimulates neovascularization in vivo (14), similar to VEGF angiogenic potential (15). Epo also participates in neuron protection from ischemic damage and cell death (16, 17). The Epo gene was fully or partially cloned from a variety of mammals (18), including the intron1/exon 2 fragment from the dolphin that also experiences hypoxic environment. All mammals display high homology as well as interspecies biological and immunological crossreactivity (18).

To counteract the possible deleterious effects of hypoxia, an immediate molecular response is initiated. This response is mediated by HIF-1, which is the mammalian master regulator of O₂ homeostasis (19, 20). HIF-1 consists of HIF-1α and HIF-1β subunits (20, 21). HIF-α is the hypoxia-inducible subunit, and its expression is regulated at both the transcription (22) and post-transcription stages (23, 24). HIF-1 is the transcription factor of many genes related to erythropoiesis (Epo, transferrin), vascularization, vasodilatation (VEGF and nitric oxide synthase), and genes involved in the glycolysis process (25). Under hypoxia, HIF-1 protein binds to a specific site in the 3′ enhancer of the Epo gene, which induces its transcription (13).

We cloned Spalax Epo and HIF-1α genes and showed dramatic differences in their expression compared with Rattus under normoxic and hypoxic conditions. Spalax Epo and HIF-1α expression pattern under hypoxia contribute to its unique mammalian record of hypoxia tolerance.

Animals. Spalax was captured in the field and housed in individual cages in the animal house of the Institute of Evolution. To test for hypoxic stress, animals were placed in a 70 × 70 × 50 cm chamber divided into separate cells. The gas mixture was delivered at 3.5 liters/min. All experiments were conducted on adults of similar weight (100–150 g). A total of 25 Spalax, from S. judaei, the Anza population, and from S. galili from the Kerem Ben Zimra population (2), and 21 laboratory Rattus norvegicus were used for Epo and HIF-1α gene quantification.

The Ethics Committee of the University of Haifa approved all experiments.

Tissues. Animals were killed by injection with Ketaset CIII (Fort Dodge, IA), 5 mg/kg of body weight. Whole organs were taken out and immediately frozen in liquid nitrogen.

RNA and cDNA Preparation. Total RNA was extracted from tissues by using TRI Reagent (Molecular Research Center, Cincinnati) following manufacturer's instructions. RNA samples were treated with DNase I (DNA-free, Ambion), and 1 μg was taken for first-strand cDNA synthesis (iScript, Bio-Rad) in a 20-μl volume. Aliquots of 1 μl of undiluted cDNA were used per PCR or real-time PCR.

Cloning of Spalax Epo (sEpo) and HIF-1α. The sEpo gene and sHIF-1α ORF were isolated by PCR using TaqDNA polymerase (Qbiogene, Illkrich, France) and oligonucleotides (Sigma Genosys, Rehovot, Israel) designed according to published human, mouse, and rat sequences. For cloning of the sEpo gene, 100 ng of genomic DNA samples from each of the four Israeli Spalax allospecies (2) was used as a template. The promoter region was amplified from two additional individuals of each species. The ORF of sHIF-1α was cloned from brain total RNA from S. galili and S. judaei. The PCR products of both genes were subcloned into pGEM T-easy vector (Promega) and transformed by electroporation into Escherichia coli XL1-blue. The sequence was determined on both DNA strands (Technion, Haifa, Israel), and analyzed by using gcg software.

Gene Quantification. Absolute gene quantification was performed by using abi prism 7000 (Applied Biosystems). Both HIF-1α and Epo gene expression were normalized to cyclophilin (26) as an internal housekeeping gene control. We quantified cyclophilin, 18-s rRNA,and β-actin in α-³²P-labeled cDNA from normoxic and hypoxic kidneys from Rattus and Spalax to ensure an equal amount of template in the real-time PCR. Both Spalax and Rattus expressed similar amounts of cyclophilin, which did not respond to hypoxia. For Spalax and Rattus gene expression comparison, we designed primers in the conserved regions of the genes by using primer express 2 software (Applied Biosystems). Cyclophilin and HIF-1α primers were identical for Spalax and Rattus. The Epo primers were not completely homologous, but amplified the same amplicon in Spalax and Rattus.

For the standard curves, dilutions of plasmid-DNA constructs containing the amplicons of each gene were used. Gene expression rates are given in copies of cDNA starting from 50 ng of total RNA. Reactions were performed by using SYBR green PCR Master Mix (Applied Biosystems) in a total volume of 25 μl. Dilutions of cDNA were used to verify efficiency of the PCR (slope = 3.3 ± 0.1, R² ≈1). The PCR plate was incubated at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Samples were isolated from individual animals, and each sample was tested in duplicate or triplicate. Unless otherwise specified, the expression of Epo and HIF-1α in Spalax represents the results obtained from both S. galili and S. judaei.

Epo Promoter Constructs. The pX2 luciferase constructs Δ18, containing 152 bp of the human Epo promoter and ΔEPΔ18, where the element named EP17 was deleted, were provided by Drs. E. Goldwasser and M. Gupta (University of Chicago, Chicago; ref. 27). To insert the Spalax-specific nucleotide substitutions in the EP17 element, point mutations were inserted by PCR, using primers with the requested substitution. In addition, Spalax promoter fragment corresponding to the human 152 bp (positions 15–167 in the Spalax gene) was amplified and inserted into pXP2 upstream to the luciferase reporter.

Transfections and Luciferase Assays. Hep3b cells were grown in DMEM (Biological Industries, Beit Haemek, Israel) containing 10% FCS. Cells were plated in six-well plates at ≈60% confluence 1 day before the transient transfection. Transfection was performed by using the following: 6 μl of FuGENE 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis) in 100 μl of serum-free media, 3.5 μg of pX2, and 0.7 μg of cmv-β-galactosidase. The reaction mix was incubated at room temperature for 20 min and added to 35-mm plates already containing adherent culture. Duplicate plates were tested; one was incubated at 37°C for 48 h under hypoxic conditions (5% CO₂/1% O₂/94% N₂), and the other under normoxic conditions (5% CO₂/95% air). For cultured cell lysis, we used M-Per mammalian protein extraction reagent (Pierce) following the manufacturer's instructions. Luciferase activity was measured by Anthos Lucy-1 (Anthos Labtech Instruments, Wals, Austria) using the Promega luciferase assay system. For β-galactosidase activity assay, we used 50 μl of cell lysis in 200 μl of 70 mM Na-phosphate buffer with 1 mM MgCl₂. The reaction mixture was incubated for 15 min in room temperature and activity was measured by Lucy-1 at OD₄₀₅. All experiments were performed in triplicate.

Data Analysis. Values are presented as mean ± SEM for n = 3–6 animals, depending on the experiment. Student's t test was used for comparison between groups (P < 0.05 considered significant).

Cloning. Epo. An Epo (sEpo) genomic fragment of ≈4.5 kb of the four Israeli Spalax allospecies was amplified and sequenced, starting at -366 bp upstream to the translation start site and ending at +1009 downstream of the translation termination site. Sequences are published in GenBank: S. galili (GenBank accession no. AJ715795), S. golani (GenBank accession no. AJ715792), S. carmeli (GenBank accession no. AJ715793), and S. judaei (GenBank accession no. AJ715794). All four Spalax species exhibit identical Epo putative peptide, apart from one nonsynonymous substitution that was found in S. golani at base pair 1912, which leads to amino acid substitution (R to Q) at position 102 of the putative peptide. A phylogenetic tree (28) of sEpo subdivided the four Spalax allospecies into two groups: the northern S. galili and S. golani, and the southern S. carmeli and S. judaei (Fig. 1). The same subdivision was found by allozymes (29), cytochrome b (30), mitochondrial DNA (31, 32), and random amplified polymorphic DNAs (33).

Fig. 1. Phylogenetic tree based on the Epo gene sequence reconstructed by using the neighbor-joining method (28).

HIF-1α. The isolated ORF of S. judaei and S. galili HIF-1α (sHIF-1α) is identical and contains 2,475 bp, coding a predicted peptide of 825 aa (GenBank accession no. AJ715791). It is highly conserved, and in comparison with the human, mouse, and rat showed ≈91% identity at the nucleotide level, and 93% identity at the amino acid level, for all three species.

Regulatory Domains in Spalax Epo. Most of the known regulatory domains in both the promoter and the enhancer of the Epo gene, including the HIF-binding site, steroid hormone receptors (DR2), and GATA site were highly conserved in Spalax compared with the human (13), mouse (34), and rabbit (35). Promising substitutions were found in the Spalax EP17 site known as a hypoxia-sensitive element (27). In humans and mice, the sequence is completely conserved: 5′-CCCCCACCCCCAC-3′. However, in Spalax, at least some of the individuals of S. judaei and S. carmeli have C-to-T substitution in position 10 in the EP17, and some of the S. golani are heterozygotes and have an A or C (M) in position 9 of the EP17. The S. galili EP17 element is identical to that of the human and mice.

Contradictory to the findings in the human modified EP17, which caused a loss of the hypoxic response (27), no significant differences were found in the hypoxic response of any of the Spalax EP17 compared with the authentic human EP17. Moreover, we did not find any difference in hypoxic activity of the Spalax promoter region compared with the equivalent human promoter. As was published (27), we found an ≈4-fold increase in luciferase reporter activity by hypoxia, suggesting that the differences in the sequence of the Spalax EP17 element or in its promoter did not affect its hypoxic inducibility.

Epo Expression. In normoxia, the levels of Epo transcript were very low, and varied between individuals in both rodents. Spalax expressed 190 ± 57 copies of Epo compared with 130 ± 53 copies of Epo in Rattus, although the differences are not statistically significant (Figs. 2 and 3).

Fig. 2. Epo gene expression in Spalax and Rattus kidneys in normoxia and after 4 h of increasing hypoxia. Values in Spalax were 190 ± 57, 6,805 ± 946, 12,449 ± 1,173, and 15,519 ± 2,208; and values in Rattus were 130 ± (more ...)

Fig. 3. Time course of Epo gene expression in Spalax and Rattus kidneys in normoxia and 10% hypoxia. The numbers of copies in 50 ng of total RNA in Spalax were 190 ± 57, 6,805 ± 946, 27,485 ± 8,322, 38,898 ± 13,548, and 3,177 ± (more ...)

Remarkably, however, in lower oxygen concentrations for 4 h (Fig. 2) and compared with normoxia, sEpo increased 82-fold (P < 0.01), significantly higher (P < 0.05) than Rattus Epo, which increased 50-fold (P < 0.05). Moreover, sEpo response is significantly and positively correlated with hypoxic stress, unlike Rattus Epo.

Because Rattus does not survive in very low oxygen concentrations (3% or 6%) for >2–6 h, we studied Epo mRNA production in 10% O₂ for different periods (Fig. 3). sEpo levels increased up to 205-fold after 24 h (P < 0.01) compared with normoxia whereas Rattus Epo levels, although significantly higher than in normoxia (P < 0.05), did not increase further after 4 h.

The Northern S. galili Is Better Adjusted to Hypoxia than the Southern S. judaei. Under 6% or 3% O₂ for 4 h or at 10%O₂ for up to 44 h, we found no differences in Epo expression among Israeli Spalax species. We then exposed S. galili (the northern species), living in heavy soil and high rainfall, and S. judaei (the southern species), living in light soil and lower rainfall, to severe hypoxia (6% O₂) for a relatively long period (10 h), conditions in which Rattus did not survive. S. galili's Epo level (Fig. 4) is 3-fold higher than in S. judaei under normoxic conditions, as well as after exposure to the hypoxic stress (P < 0.05, n = 5).

Fig. 4. Comparative Epo gene expression in S. galili and S. judaei under normoxia and 6% O2 for 10 h. S. galili values were 269 ± 65 and 10,687 ± 1,506; and S. judaei values were 85 ± 30 and 3,739 ± 1,620, under normoxia and hypoxia, (more ...)

HIF-1α Expression. We also quantified HIF-1α expression in the same cDNA kidney samples isolated from Spalax and Rattus. Under normoxic conditions, sHIF-1α levels were two times higher compared with rHIF-1α (P < 0.05; Fig. 5). Moreover, rHIF-1α does not respond to hypoxia, whereas sHIF-1α does significantly. A dramatic increase (P < 0.01) of sHIF-1α followed increased hypoxia, contrasting with rHIF-1α.

Fig. 5. HIF-1α gene expression in Spalax and Rattus kidneys in normoxia and after 4 h of increased hypoxia. Spalax values were 48,644 ± 5,407, 47,288 ± 4,357, 85,858 ± 12,745, and 102,890 ± 13,797; and Rattus values were (more ...)

At a relatively mild hypoxic stress of 10% O₂ and up to a 12-h exposure, sHIF-1α expression is similar to its levels under normoxia (Fig. 6). After exposure for 24 h or more to 10% O₂, sHIF-1α levels in kidneys decreased to one half-fold (P < 0.01) compared with normoxia, whereas rHIF-1α levels fluctuated insignificantly compared with normoxia (Fig. 6).

Fig. 6. Time course of HIF-1α expression in Spalax and Rattus kidneys in normoxia and 10% hypoxia. Spalax values were 48,644 ± 5,407, 47,288 ± 4,357, 46,599 ± 3,302, 24,832 ± 2,910, and 34,041 ± 1,248 and Rattus (more ...)

In most of the experiments, we did not find significant differences in HIF-1α expression among the Spalax species that were tested. Only at extreme conditions, after 10 h at 6% O_2, HIF-1α mRNA levels were 40% higher in the northern species S. galili compared with the southern species S. judaei.

General Overview. All higher organisms developed adaptive mechanisms to detect and respond to hypoxia. However, animals that chronically inhabit hypoxic ecological niches developed uniquely effective strategies to survive under hypoxia. High-altitude mammals (36), diving mammals (37), and particularly Spalax (38) that represent an extreme case among subterranean mammals (1), faced hypoxia and developed adaptations to tolerate it. Higher muscle myoglobin, larger blood buffer capacity, and hemoglobin properties allow these animals better O₂ utilization (8, 38). Nevertheless, Spalax differs dramatically from all diving mammals that are exposed intermittently to hypoxia, recharging O₂ breathing (38). High-altitude mammals are often limited in their vertical ascent and hypoxic exposure. Spalax, by contrast, spends its life mostly underground in sealed burrows (1, 2), and experiences hypoxia down to 7% O₂ (I.S., A.A., and E.N., unpublished work), and probably much lower when the burrows are flooded by rainwater. Moreover, at least three of the allospecies of Spalax studied here, the S. galili, S. golani, and S. carmeli (2) inhabit heavy soil and high rainfall, drastically limiting soil ventilation and gas permeability (1). Consequently, Spalax is confronting the hypoxia primarily during its winter activities (1) under persistent hypoxia.

A common adaptive strategy of mountaintop and diving mammals is the down-regulation of metabolism (39), i.e., myocardial and skeletal muscle contractile activity (36–40). However, permanent activity under hypoxia necessitates constant O₂ delivery to tissues, especially muscles used in burrowing. Erythrocyte supply is critical in hypoxia, depending on Epo expression (41) and its regulation by HIF-1 (25); hence, our analysis of these two genes in Spalax.

Epo Is a Key Factor to Confront Hypoxia. Only partial cloning of dolphin Epo was heretofore reported in mammals living under hypoxia (18). Therefore, we present here the first comprehensive study, to our knowledge, on Epo and HIF-1 gene expression in an animal chronically exposed to hypoxic stress.

Erythropoesis is regulated by several cytokines, initiated by bone marrow pluripotent stem cells, and terminating in mature erythrocytes. Epo acts primarily on colony-forming unit erythroid proliferating into mature erythrocetes (41, 42). Likewise, Epo participates in angiogenesis (14, 15), and stimulates postnatal neovascularization (43). Epo is synthesized mainly in fetal liver (11), shifting to the kidneys shortly after birth (12). Nevertheless, Epo expression has been detected also in the lung, spleen, brain, and testis (44).

Epo synthesis in hypoxia is regulated at the mRNA level (45, 46) and is controlled by both transcription and posttranscriptional mechanisms (47). Decreased oxygen tension in tissues (in anemia or hypoxia) induces glycolytic gene activity, growth factors, and hormones (20). Transcription of most of these genes is induced by HIF-1, the main regulator of oxygen homeostasis in mammals (19). In Epo, HIF-1 binds to a specific site in the enhancer at the 3′ end of the gene (25). HIF-1 is a heterodimer composed of two subunits, both of which are basic helix–loop–helix proteins in the PAS (Per-AHR-ARNT-SIM) family of transcription factors. The HIF-1α subunit responds to hypoxia, and HIF-1 activation is correlated with oxygen-dependent accumulation of HIF-1α protein (20).

Spalax Epo Expression Is Adapted to Oxygen Fluctuations. The Epo gene of all Israeli Spalax contains all of its known regulatory sites (13). The EP17 element is involved in hypoxia-regulated Epo gene expression (27). Transfections of Hep3B cells with the minimal active fragment of the human promoter, containing the EP17 element, conferred a 4-fold induction in luciferase-reporter activity due to hypoxia. Internal deletion or multiple point mutations in the EP17 resulted in loss of response to hypoxia, suggesting that factors binding to the EP17 element activate Epo transcription in cells under hypoxia. We have found that the EP17 element in three of the four Israeli Spalax species contain one nucleotide substitution compared with the human (27) and mouse (12). However, these changes do not affect Spalax EP17 response to hypoxia, which resembles the human's. Notably, demolishing the hypoxic response of the human EP17 element (27) involved more than one nucleotide substitution, and the sequence of the rabbit EP17 element (35) is different from that of the human, mouse, and Spalax, yet is still active. Furthermore, the Spalax authentic equivalent promoter fragment also induced hypoxic responses similar to the human homolog. Therefore, we conclude that any changes in sEpo expression under hypoxic stress cannot be attributed to the changes found in its promoter as compared to other species.

Rattus kidney responds to hypoxia by increasing Epo production (45, 48). We found that maximum Epo expression in the Rattus kidney is 50-fold higher compared with its normoxic levels after 4 h at 6% O₂. Spalax, by contrast, shows a unique, extreme ability to respond to long-term hypoxia, and maximal expression, found at 10% O₂ after 24 h, is five times higher than the maximal expression of Rattus Epo (Figs. 2 and 3). However, after 44-h hypoxia at 10% O_2, Epo levels in Spalax kidneys decreased and were similar to the Rattus Epo levels under identical conditions. It has been shown that the injection of recombinant human Epo to Rattus did not affect Epo production under hypoxia; thus, this decline is not due to feedback inhibition (49). Nevertheless, others reported (50) that after 24 h of hypoxia, Epo protein level reaches its maximum, then significantly decreases, and returns to normoxic values after 14 days. However, the number of colony-forming unit erythroid cells, the target cells of Epo (41), reach a peak on day 14 and then progressively decrease. Therefore, it can be suggested that the increase in Epo during the first 24 h of hypoxia, similar to what we noticed in Spalax Epo mRNA, is sufficient to induce a satisfactory rate of erythropoiesis for the maintenance of a steady state under longer hypoxic conditions.

Because Rattus did not survive at lower oxygen concentrations for more than 4–6 h, we exposed animals to different oxygen concentrations (10%, 6%, and 3% O₂) for 4 h. It should be reemphasized that Spalax, in contrast to Rattus, demonstrated no deleterious effects and no changes of behavior under 6% or 3% O₂ for up to 14 h when the observations were terminated. The data suggest that Rattus Epo does not respond significantly to different O₂ concentrations. Hence, Rattus cannot adapt to increased hypoxic stress by increasing Epo production. Spalax, in contrast, responds according to the stress in an oxygen-concentration-dependent manner, and Epo levels after 4 h are maximal at the lowest O₂ supply studied (3%); i.e., it is adapted to live in a wide range of oxygen levels and responds appropriately to variable oxygen stresses. The quick and stress-dependent elevation in Epo mRNA levels in Spalax support its exposure to abrupt, drastic falls in oxygen supply, down to a low % O₂, in its sealed tunnels while flooded during rain. In this situation, when the animal is provoked into heavy work, exhausting the already limited supply of oxygen present, erythrocyte production is induced to better use the limited levels of available oxygen.

Epo was shown to have nonerythropoietic functions as well (reviewed in ref. 51). It protects brain neurons and the spinal cord from ischemic damage in animal models; it plays a role in the modulation of the ventilatory acclimatization to hypoxic exposure; it can reduce the infarct size in patients after ischemic brain stroke; and it protects the retina from experimentally light-induced retinal degeneration. Therefore, considering the fact that Epo-dependent erythropoiesis takes a few weeks to elevate the hematocrit (52), and as Spalax hematocrit is higher than in other rodents (7), the Epo mRNA pattern of expression in Spalax might have nonerythropoietic protective functions to meet its hypoxic environment.

Moreover, we observed higher levels of Epo and HIF-1α mRNA in the northern allospecies S. galili compared with the southern S. judaei species under both normoxic conditions and after long periods (10 h) of severe hypoxia (6% O₂). These results, reflected also in the phylogentic tree of Epo that separates the northern from the southern species, complement previous observations on higher hematocrit and Hb levels in the northern species (7) and its higher genetic tolerance to lower O₂ levels (3). This finding supports our hypothesis of adaptive respiratory variations associated with climatic and consequent hypoxic severity, also within the Spalax ehrenbergi superspecies.

sEpo Response to Hypoxia Is Different from Its VEGF Response. The Epo-adaptive expression patterns in Spalax are different from those of VEGF (4). In Spalax muscle, the most energy consuming tissue, VEGF is maximal in normoxia with no further increase in hypoxia. In Rattus, VEGF is significantly lower than Spalax in normoxia, but increases 2.2-fold in hypoxia (4). VEGF is needed for relatively longer processes, such as neovascularization and angiogenesis, whereas Epo is mainly used for relatively shorter processes of maintaining the erythrocyte level and urgent increases of erythrocyte production in hypoxia. Therefore, in an animal like the Spalax, which spends most of its life underground exposed to drastic fluctuations in oxygen levels, VEGF constitutively supports long-term neovascularization, whereas Epo levels respond quickly to the hypoxic stress.

HIF-1α Levels in Spalax Rise Under Severe Hypoxia. Previous reports (22) on the in vivo regulation of HIF-1α expression by hypoxia are contradictory. HIF-1α mRNA concentrations of mice exposed to 0.1% carbon monoxide for 4 h were not up-regulated despite a strong serum Epo protein induction. However, a nonquantitative Northern hybridization study on rats exposed to 7% O₂ for 60 min before sacrifice, indicated a modest increase of HIF-1α mRNA levels, but returned to baseline after 4 h of (23). Our results show that the levels of HIF-1α in Rattus kidneys did not change significantly under different hypoxic conditions compared with its normoxic levels. It seems that HIF-1α mRNA (22) and protein (24) response to hypoxia is transient and short. Our findings indicate that the induction of Rattus Epo expression due to hypoxia is probably not dependent on an increase of HIF-1α mRNA level in kidneys, but induced either by the existing HIF-1 present in the kidney under normoxia or secreted by other organs and/or by posttranscriptional mechanisms (47). The inability of the Rattus kidney to respond to hypoxia by elevated HIF-1α mRNA level (Figs. 5 and 6) may be related to our findings that Rattus kidney levels of Epo are significantly lower compared with Spalax under different hypoxic stresses and do not change significantly with time length or the degree of the hypoxic stress (Figs. 2 and 3). We can assume that the inability of Rattus HIF-1α to respond to the hypoxic stress for periods >60 min (23), and its Epo unresponsiveness to the duration or the degree of the hypoxic stress, contribute to its death under severe hypoxia and after a relatively short time.

Spalax kidneys, in contrast to Rattus, contribute to HIF-1α gene expression, first by maintaining a high level in normoxia, and second, by stimulating its mRNA synthesis in severe hypoxia. Thus, Spalax seems to have more than one mechanism of HIF-1α activity that supports its tolerance to hypoxia: (i) Spalax has a 2-fold higher level of kidney HIF-1α in normoxia compared with Rattus; (ii) the up-regulation of Spalax Epo levels under a long period of mild hypoxic conditions (10% O₂) is controlled through the induction of high levels of HIF-1 present at basic normoxic conditions and/or by posttranscriptional processes in Epo (48) and HIF-1α (20, 21); (iii) under severe hypoxia, Spalax HIF-1α expression in the kidneys, the main Epo synthesis organ, is elevated in a stress-related mode, although the increase at O₂ lower than 6% O₂ is not significant (Fig. 5). We hypothesize that at 6% O₂ Spalax HIF-1α reaches its maximal efficiency and/or capacity to respond to the hypoxic stress. Nevertheless, this occurrence does not affect its ability to survive at 3% O₂.

It should be mentioned that in vitro (24) HIF-1α protein was not detected in the nuclei of HeLaS3 cells under normoxic conditions. However, exposure of the cells to very low O₂ (0–5%) for periods of 2 min to 4 h revealed synthesis of nuclear HIF-1α protein that was observed after 2 min and continued to accumulate for up to 60 min. At 60 min, it reached a maximum level that was maintained for up to 4 h of hypoxic exposure. Noteworthy, we must emphasize that we have noticed (I.S., A.A., and E.N., unpublished work) significant differences in the pattern of expression of HIF-1α as well as VEGF mRNAs ex vivo compared with in vivo. Furthermore, a special emphasis in our future studies should be addressed to the expression of HIF-2α. This emphasis is in light of results (53) that indicate a selective hypoxic induction of HIF-1α and -2α and their possible dependent target gene activation in different parts of the kidney. Accordingly, peritubular cortical fibroblasts were identified as the site of both Epo and HIF-2α production, suggesting a possible role for HIF-2α in Epo regulation.

Accumulation of sHIF-1α and sEpo shown here and in eryth-rocyte maturation (50, 54), may indicate a cascade-like kinetics: HIF-1α, the hypoxia-sensitive subunit of HIF-1 that induces Epo production, reaches its maximal expression in Spalax kidney at 12 h. Epo expression, regulated by the binding of HIF-1 protein to its promoter, lags and achieves maximal level in Spalax kidney at 24 h. Maximal erythrocyte maturation, driven by Epo protein, occurred when Epo levels decreased (50, 54).

Prospects. A few questions are raised in this study and need further investigation: (i) what is the role of Spalax Epo mRNA stability in the observed high levels after long-term hypoxia? Northern blot analyses showed that the steady state of Epo mRNA levels in Hep3B cells increase >50-fold in response to hypoxia, but nuclear runoff experiments demonstrated only a 10-fold increase in Epo gene transcription under identical conditions. This finding suggests that the stability of Epo mRNA and its steady-state levels are modulated by both transcriptional and posttranscriptional events (42); (ii) is the HIF-1α promoter also induced by hypoxia and does it contribute to the HIF-1α gene expression in Spalax? At present, there is no evidence for HIF-1α promoter's response to hypoxia (24). It is of great interest to test Spalax Epo and HIF-1α and -2α expression, at both mRNA and protein levels, in fluctuating and severe hypoxia and in hypoxia followed by reoxygenation, because Spalax lives in a greatly fluctuating oxygen atmosphere, ranging from very low to normal oxygen levels.

Clearly, Epo and HIF-1α are only two of the genes in Spalax's gene battery of hypoxic tolerance adaptation that evolved during its 40 million years of underground evolution (1). Spalax is undoubtedly an extreme wild mammal model exposed sporadically to extreme levels of hypoxia, particularly during heavy winter rains. The adaptive trajectory of Spalax evolved toward ever-increasing hypoxia tolerance rather than the alternative mammalian channel of hibernation.

Future studies in our laboratory aim at probing the extensive adaptive battery of genetic responses of Spalax to variable hypoxic stress through comprehensive functional genomic studies and to unravel the numerous genes collaborating in Spalax hypoxia tolerance. These studies could later be used in human gene therapy in the fight against ischemia (55) and cancer and contribute to life in environmental extremes such as outer space flights and deep sea diving.

We thank Drs. E. Goldwasser and M. Gupta for providing the human Epo promoter constructs; Drs. I. Vlodavsky, K. Skorecki, A. Levy, and their laboratory staff (Technion) for their excellent technical assistance; Prof. M. Gassmann (University of Zurich, Zurich) and Dr. D. Neumann (Tel-Aviv University, Tel Aviv) for critical review of the manuscript; Integrated DNA Technologies (Coralville, IA); and Drs. S. Ross and R. Devor for the gene quantification training course (A.A.). This work was supported by the Ancell–Teicher Research Foundation for Genetics and Molecular Evolution. This work is a partial fulfillment of requirements for a Ph.D. (I.S.).