Isoform-Specific Expression of Hypoxia-Inducible Factor-1{alpha} During the Late Stages of Mouse Spermiogenesis

Hugo H. Marti1, Dörthe M. Katschinski, Klaus F. Wagner, Leonhard Schäffer, Bettina Stier and Roland H. Wenger

Max-Planck-Institute for Physiological and Clinical Research (H.H.M.), Department of Molecular Cell Biology, D-61231 Bad Nauheim, Germany; Institute of Physiology (D.M.K., K.F.W., B.S., R.H.W.) and Clinic of Anaesthesiology (K.F.W.), Medical University of Lübeck, D-23538 Lübeck, Germany; and Department of Obstetrics and Gynaecology (L.S.), University Hospital Zürich, CH-8091 Zürich, Switzerland

Address all correspondence and requests for reprints to: Roland H. Wenger, Ph.D., Carl-Ludwig-Institut für Physiologie, Universität Leipzig, Liebigstrasse 27, D-04103 Leipzig, Germany. E-mail: wenr{at}medizin.uni-leipzig.de


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 REFERENCES
 
The heterodimeric hypoxia-inducible factor (HIF)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homeostasis, including erythropoietin, vascular endothelial growth factor, and factors involved in glucose transport and metabolism. The mouse Hif1a gene is expressed from two distinct promoter/first exon combinations resulting in tissue-specific (mHIF-1{alpha}I.1) and ubiquitous (mHIF-1{alpha}I.2) mRNA isoforms. By in situ hybridization, we detected mHIF-1{alpha}I.1 mRNA exclusively in the elongated spermatids of the testis. In vitro studies indicated that the switch from mHIF-1{alpha}I.2 to mHIF-1{alpha}I.1 mRNA expression does not occur at the premeiotic stages of mouse spermatogenesis. Exposure of mice to hypoxic conditions induced mHIF-1{alpha}I.2 protein in spermatocytes and probably in Sertoli cells but not in spermatogonia. In contrast, expression of the putative mHIF-1{alpha}I.1 protein in spermatozoa of the testis and epididymis was oxygen independent and located to the midpiece of the spermatozoal flagellum. Both the switch in transcript expression during spermiogenesis and the unexpected protein localization in mature sperm cells suggest a so far unrecognized function of HIF-1{alpha}.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 REFERENCES
 
IN MOUSE TESTIS, spermatogenesis is a highly ordered differentiation process, involving profound transcriptional and morphological changes. Spermatogenesis is initiated by the differentiation of self-replicating spermatogonia at the basal layers of the seminiferous tubuli to become committed intermediate spermatogonia. After two further mitotic divisions, primary spermatocytes are formed which undergo the two rounds of meiotic divisions resulting in haploid round spermatids. In a process called spermiogenesis, these round spermatids mature via elongated spermatids to spermatozoa, which are finally released into the lumen of the seminiferous tubuli. Spermatogenesis appears in synchronized waves of layers of differentiating germ cells that sequentially migrate from the basal toward the luminal regions of the seminiferous tubuli, allowing to distinguish stages I to XII of the cycle of spermatogenesis (reviewed in Ref. 1).

During nuclear elongation of haploid spermatids, the rate of transcription declines and becomes undetectable in elongated spermatids. Nevertheless, ongoing translation of a number of sperm cell-specific structural proteins and isoforms of metabolic enzymes is required for the production of spermatozoa. Therefore, specific mRNA isoforms containing long poly(A) tails are stored as translationally inactive ribonucleoprotein particles. In transcriptionally inactive states, these mRNA isoforms are recruited into translationally highly active polysomes to ensure ongoing protein synthesis (reviewed in Refs. 2, 3, 4). Prominent examples include the nuclear transition proteins (5, 6) and later in spermiogenesis the protamines (7, 8, 9), which replace the histones and lead to compaction of the chromatin.

There are also various testis-specific isoforms of glycolytic enzymes, which are expressed in the haploid stages of spermatogenesis and which are still active in mature spermatozoa, including phosphoglycerate kinase 2 (10, 11), glyceraldehyde 3-phosphate dehydrogenase-2 (12), and lactate dehydrogenase C (13). Because of the greater oxygen diffusion distance, the luminal regions of the seminiferous tubuli as well as the epididymis are likely to be hypoxic when compared with the basal regions of spermatogonial self-renewal. In addition, the high proliferative capacity of the germinal epithelium suggests a pronounced oxygen consumption, thereby further decreasing the oxygen concentration. Thus, testis-specific glycolytic enzyme isoform expression might be related to hypoxic adaptation by altered anaerobic energy metabolism. Indeed, sperm capacitation, motility changes, acrosome reaction, and fertilization is exclusively dependent on anaerobic glycolysis and can occur under strictly anaerobic conditions (14). However, the influence of oxygen concentration, consumption, and metabolism on the molecular events during spermatogenesis, spermatozoa release, in ejaculated sperm, and during fertilization is largely unknown.

The hypoxia-inducible factor 1 (HIF-1) is an ubiquitously expressed transcriptional master regulator of many genes involved in mammalian oxygen homeostasis, including the hormones and related compounds erythropoietin, vascular endothelial growth factor, inducible nitric oxide synthase, and heme oxygenase-1 (reviewed in Refs. 15, 16). HIF-1 is also a major regulator of the glycolytic capacity (17). HIF-1 is a {alpha}1ß1 heterodimer specifically recognizing the HIF-binding site within cis-regulatory hypoxia response elements. Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein targets the HIF-1{alpha} subunit for rapid ubiquitination and proteasomal degradation (18). The von Hippel-Lindau tumor suppressor protein binding requires oxygen-dependent prolyl hydroxylation of HIF-1{alpha} (19, 20). We previously cloned the mouse HIF-1{alpha} gene (Hif1a) and found that its expression is driven by two different promoters located 5' to two alternative first exons designated exon I.1 and exon I.2 (21, 22). Whereas the upstream exon I.1 promoter exhibits tissue-specific features, the downstream exon I.2 promoter is a typical housekeeping-type promoter driving ubiquitous transcription (22). The mouse mHIF-1{alpha}I.1 mRNA isoform encodes for a predicted protein product that is 12 amino acids shorter than the predicted mHIF-1{alpha}I.2 protein. Despite its vicinity to the basic-helix-loop-helix DNA binding domain, this N-terminal deletion does not affect DNA binding efficiency (23). To define the cell types expressing the tissue-specific mHIF-1{alpha}I.1 mRNA, in situ hybridization of the two HIF-1{alpha} mRNA isoforms was performed in mouse tissues.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 REFERENCES
 
mHIF-1{alpha}I.1 mRNA Expression Is Confined to Spermatids of Distinct Developmental Stages
Antisense in situ hybridization probes specific for the unique first exons of the two mouse HIF-1{alpha} mRNA isoforms were used to analyze their cellular expression patterns in vivo. As expected from previous RNase protection (22) and in situ hybridization (24) studies, the mHIF-1{alpha}I.2 main mRNA isoform probe yielded faint to moderate ubiquitous signals throughout all organs that were clearly above the controls obtained with the sense probe. The mHIF-1{alpha}I.1 mRNA isoform was undetectable in brain, liver, kidney, heart, lung, and spleen (data not shown). However, when adult mouse testis was analyzed, a strong, specific signal could be observed in some but not all seminiferous tubuli using the antisense mHIF-1{alpha}I.1 probe. In contrast, the mHIF-1{alpha}I.2 antisense probe revealed an ubiquitous expression pattern localized to the epithelium of all seminiferous tubuli. As before, the sense probes did not result in detectable signals on adjacent slices (Fig. 1AGo). At higher magnification, it became apparent that hybridization with the mHIF-1{alpha}I.1 but not with the mHIF-1{alpha}I.2 antisense probe was dependent on the stage of the cycle of the seminiferous epithelium (Fig. 1BGo).



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Figure 1. Localization of the HIF-1{alpha} mRNA Isoforms mHIF-1{alpha}I.1 and mHIF-1{alpha}I.2 in Adult Mouse Testis

In situ hybridization on sections of adult mouse testis was performed using 35S-labeled antisense and sense probes specific for the two alternative first exons (I.1 and I.2) of mouse HIF-1{alpha}. A, Dark field; original magnification, 25x; scale bar length, 1 mm. B, Dark field (rows 1 and 2); bright field (row 3); original magnification, 100x; scale bar length, 250 µm. C, Bright field (rows 1 and 2, left part); dark field (row 2, right part); original magnification, 400x; scale bar length, 62.5 µm.

 
A closer inspection at high magnification in the bright field (Fig. 1CGo) allowed the detection of silver grains over elongated but not round spermatids located to the most luminal layers of germ cell differentiation. Luminal localization and the morphological pattern of cell association suggest that the mHIF-1{alpha}I.1 mRNA isoform is expressed in those segments of the mouse seminiferous tubuli that correspond to the developmental stages VI to VIII of the cycle of the seminiferous epithelium. At these stages, the spermatids are in steps 15 and 16 of spermiogenesis just before the release of mature spermatozoa (1). Again, a comparatively weaker signal could be seen with the mHIF-1{alpha}I.2 antisense probe, which appears primarily in a region overlapping with the localization of Sertoli cells and the basal germ layers of the seminiferous epithelium rather than in the round spermatids. Thus, these results indicate a switch from mHIF-1{alpha}I.2 to mHIF-1{alpha}I.1 mRNA isoform expression during or after meiosis.

Evidence that Induction of the Testis-Specific mHIF-1{alpha}I.1 mRNA Isoform Starts in Late Post-Meiotic Germ Cells
Four different cell lines corresponding to Leydig cells (TM3), Sertoli cells (TM4), spermatogonia (GC-1 spg), and spermatocytes (GC-2 spd(ts)) were chosen for expression studies. These cells were exposed to hypoxic conditions (1% O2) for 4 h and analyzed by immunoblotting. For comparison, three non-testis- derived cell lines, HeLa cervic carcinoma, MCF-7 adenocarcinoma, and Jurkat T cell leukemia, were treated accordingly. As shown in Fig. 2AGo, HIF-1{alpha} protein was strongly induced by hypoxia in nuclear extracts isolated from all cell lines. When cultured under hypoxic conditions, the levels of HIF-1{alpha} in the testis-derived cell lines were clearly higher than the levels in the non-testis-derived cell lines. Compared with HIF-1{alpha}, the differences in the levels of HIF-1ß/ARNT under hypoxic conditions were less pronounced among the cell lines analyzed. Under normoxic conditions, the levels of ARNT were decreased in nuclear extracts of some of the cell lines, despite the fact that ARNT is normally expressed in a constitutive manner. We previously showed that, when HIF-1{alpha} is absent, ARNT is decreased in normoxic nuclear extracts due to leakage out of the nucleus during preparation rather than to a true oxygen-dependent regulation (25). The signals obtained with an antibody derived against an unrelated transcription factor (Sp-1) confirmed that similar protein amounts were loaded in each lane.



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Figure 2. Expression Analysis of HIF-1{alpha} in Testis-Derived Cell Lines

A, Immunoblot analysis of Leydig (TM3), Sertoli (TM4), GC-1 spg spermatogonia (GC-1), and GC-2 spd(ts) diploid spermatocyte/haploid spermatid (GC-2) as well as HeLa, MCF-7, and Jurkat control cell lines exposed to normoxia (-) and hypoxia (+) for 4 h. B, RT-PCR analysis of mHIF-1{alpha}I.1 and mHIF-1{alpha}I.2 mRNA expression in the testis-derived cell lines. C, Representative luciferase reporter gene assay with exon I.1-specific and exon I.2-specific promoters under normoxic (open columns) or hypoxic (filled columns) conditions.

 
Because to date there is no antibody available that can distinguish between the different N-termini of the two mouse HIF-1{alpha} protein isoforms, specific RT-PCR was used to show that both HIF-1{alpha} mRNA isoforms are simultaneously expressed in the four testis-derived cell lines (Fig. 2BGo). The amount of PCR products cannot be directly compared between the two products because they encompass G + C poor (I.1) and G + C rich (I.2) regions, respectively. However, the signal intensity did not significantly differ between the cell lines and remained unaffected by hypoxic exposure. As expected from these data, luciferase reporter gene experiments using exon I.1 (21) and exon I.2 (22) 5' flanking DNA fragments showed that the exon I.1 and I.2 promoter activities remained largely unaltered between these cell lines when compared with a promoterless construct (Fig. 2CGo). Neither exposure to hypoxic conditions (Fig. 2CGo) nor induction of differentiation by treatment with testosterone at up to 1 µM and/or FSH at up to 0.5 U/ml (data not shown), significantly altered the reporter gene activities in the four cell lines.

Initially, the GC-2 spd(ts) cell line has been shown to consist of both diploid spermatocyte and haploid early spermatid-like cells (26), but haploid cells could not be detected in later studies (27). Nevertheless, our in vitro results indicate that a change in transcriptional activity does not occur during the premeiotic stages of spermatogenesis and hence cannot explain the strong expression of the I.1 mRNA isoform and the complete lack of the I.2 mRNA isoform in the haploid mature spermatids as observed by in situ hybridization. Thus, the I.1 mRNA isoform accumulation might be due to a change in transcriptional activity at the postmeiotic stages of spermiogenesis that, however, cannot be investigated in cell culture models in vitro.

Hypoxia Induces HIF-1{alpha} Protein Expression in Spermatocytes
To investigate oxygen-regulated HIF-1{alpha} protein expression in testis, adult male mice were exposed to hypobaric hypoxia for 6 h, and the testes were analyzed by indirect immunohistochemistry. As shown in Fig. 3AGo, under normoxic conditions we were able to detect moderate HIF-1{alpha} expression that is induced under hypoxic conditions in a region overlapping with Sertoli cells and spermatocytes. HIF-1{alpha} is mainly expressed in the nuclei of pachytene spermatocytes (as characterized by their large nuclei) and to a lesser extent in the adjacent round spermatids (Fig. 3BGo). In contrast, HIF-1{alpha} was undetectable in the nuclei of the basal spermatogonial layer and the innermost layer of mature elongated spermatids (Fig. 3BGo). Control experiments using the secondary antibody alone indicated that the weak staining of the matrix surrounding the seminiferous tubuli but not the cellular staining was due to unspecific antibody binding (Fig. 3Go, A–C). Surprisingly, there was also a positive HIF-1{alpha} protein staining in the lumen of the seminiferous tubuli where the spermatozoal tails are located (Fig. 3Go, B and C). Under higher magnification, it became apparent that hypoxia-inducible HIF-1{alpha} protein is located to part of the flagellum adjacent to the head rather than to the typically shaped end-stage spermatid nuclei (Fig. 3DGo). Thus, the spermatid-specific mHIF-1{alpha}I.I mRNA isoform might give rise to a protein isoform that occupies a distinct cellular compartment of mature spermatozoa.



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Figure 3. Immunohistochemistry of HIF-1{alpha} in Mouse Testis

Adult control mice (20% oxygen) or mice exposed to hypobaric hypoxia (corresponding to 6% oxygen) for 6 h were analyzed. For controls, the primary polyclonal IgY antibody was omitted. A, Original magnification, 100x; scale bar length, 200 µm. B, Filled and open arrowheads indicate pachytene spermatocytes and spermatogonia, respectively. Original magnification, 200x; scale bar length, 100 µm. C, Original magnification, 400x; scale bar length, 50 µm. D, Filled and open arrowheads indicate the elongated nuclei and midpiece of the flagellum, respectively, of spermatozoa. Original magnification, 1000x; scale bar length, 20 µm.

 
HIF-1{alpha} Protein Is Expressed in the Midpiece of the Flagellum of Mouse Epididymal Spermatozoa Independent of the Oxygen Concentration
We next analyzed whether expression of the mHIF-1{alpha}I.I protein isoform persisted in mature spermatozoa. As shown in Fig. 4Go, A–C, HIF-1{alpha} was still expressed in the spermatozoa of the epididymis located adjacent to the head that does not contain HIF-1{alpha} protein. HIF-1{alpha} protein was also expressed in the epithelial basal layer of the epididymis, which might be attributed to the ubiquitous mHIF-1{alpha}I.2 isoform (Fig. 4BGo). Staining of the surrounding smooth muscle layers was due to unspecific antibody binding (Fig. 4Go, A and B).



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Figure 4. Immunohistochemistry of HIF-1{alpha} in Mouse Epididymis

Mice were treated as described in Fig. 3Go. A, Original magnification, 100x; scale bar length, 200 µm. B, Original magnification, 200x; scale bar length, 100 µm. C, Original magnification, 1000x; scale bar length, 20 µm.

 
To better define the subcellular localization of HIF-1{alpha}, spermatozoa were isolated from the epididymis and vas deferens, exposed to normoxic or hypoxic conditions for 4 h, and examined by immunofluorescence using a polyclonal IgY antibody (28) (Fig. 5AGo) or a monoclonal antibody (29) directed against another epitope of HIF-1{alpha} (Fig. 5BGo). Both antibodies, but not the secondary antibody alone, detected abundant HIF-1{alpha} protein in the midpiece of the flagellum of mouse spermatozoa under normoxic and hypoxic conditions.



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Figure 5. Subcellular Localization of HIF-1{alpha} in Mouse Spermatozoa

Immunofluorescence analysis of spermatozoa isolated from epididymis and vas deferens after in vitro exposure to 20% or 1% oxygen for 4 h. A polyclonal chicken IgY antibody (A) or the monoclonal mouse H1{alpha}67 IgG2b antibody (B) were applied. For controls, the samples were treated with the corresponding secondary antibodies alone. Original magnification, 630x; scale bar length, 50 µm.

 

    Discussion
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 REFERENCES
 
Testis-specific transcription factor isoform expression is not an uncommon finding. For example, expression of testis-specific promoter/first exon combinations of the TATA-binding protein (TBP) (30) and GATA-1 (31) or testis-specific alternative splicing of cAMP responsive element modulator {tau} (32) have been reported. TBP mRNA is stored as messenger ribonucleoprotein for up to 1 wk, after which it is required for the transcription of sperm-specific proteins (e.g. protamines) under conditions when general transcription ceases (2). mRNA expression of the testis-specific TBP and cAMP responsive element modulator {tau} isoforms begins in early round spermatids, and the corresponding proteins are no longer detectable in mature spermatozoa.

In marked contrast, the detection of the mHIF-1{alpha}I.1 mRNA isoform in the head of mature haploid spermatids and of the HIF-1{alpha} protein in the midpiece of the flagellum of spermatozoa represents to our knowledge the first example of testis-specific transcription factor expression beyond the release of spermatozoa from the seminiferous tubuli. To date, the function of HIF-1{alpha} at this specific site is unclear. Intriguingly, many HIF-1 target genes, especially the glycolytic enzymes, are also expressed in the testis as specific isoforms (10, 11, 12, 13). However, because the spermatozoal nuclei are thought to be transcriptionally inactive, it seems unlikely that HIF-1 functions as a transcription factor in sperm. Rather, the high levels of HIF-1{alpha} might be required in oocytes after fertilization that occurs in a hypoxic environment. Because mice containing a null mutation in the second exon of the Hif1a gene are nonviable (33), an exonI.1-specific mouse knockout model will be required to elucidate the functional significance of specific HIF-1{alpha} isoform expression during mouse spermiogenesis.

Another intriguing question is the exact mechanism of the mHIF-1{alpha}I.1 mRNA accumulation in the differentiated spermatids. Our results with in vitro cultured spermatogonia and spermatocyte cell lines indicate that the exon I.1 promoter does not become activated during spermatogenesis in these premeiotic stages. Interestingly, the proximal exon I.1 promoter contains an open-reading frame for a so-called premordial peptide (21). These repetitive elements have been reported to be transcribed in the sex-determining region of the Y chromosome (34). While the mouse Hif1a gene is not located on the Y chromosome, this sequence might be responsible for activating the exon I.1 promoter during male postmeiotic spermiogenesis. Clearly, in vitro transcription assays and transgenic mouse models are needed to clarify this point.

While exposure to hypoxia induced mHIF-1{alpha}I.2 protein levels in the testis, it did not further induce mHIF-1{alpha}I.1 protein in epididymal spermatozoa or after in vitro exposure of isolated spermatozoa. The most obvious explanation for this effect might be the lack of proteasomal activity in mature spermatozoa, rendering mHIF-1{alpha}I.1 protein stability already under normoxic conditions. Although we cannot formally exclude that the mHIF-1{alpha}I.2 protein corresponds to the isoform detected in spermatozoa, the switch from mHIF-1{alpha}I.2 to mHIF-1{alpha}I.1 mRNA isoform expression during spermatogenesis and the complete lack of detectable mHIF-1{alpha}I.2 mRNA in spermatids strongly suggest that the mHIF-1{alpha}I.1 mRNA is translated in postmeiotic spermatids. The definitive proof would require antibodies specific for the two different predicted N-termini (the actual N-termini are unknown for both isoforms and might differ from the prediction due to posttranslational processing). However, as we did not find any functional differences of the two isoforms in vitro (23), the biological significance of our findings probably lies in the fact that an alternative promoter is active to ensure the expression of HIF-1{alpha} at unusually late stages of spermiogenesis, rather than the expression of a structurally or functionally different protein isoform.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
 REFERENCES
 
Animal Experimentation
Animal experiments were performed in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the local Governmental Commissions for the Care of Animals.

In Situ Hybridization
The technique used for in situ hybridization was essentially as described by Breier et al. (35). A cDNA clone containing mouse Hif1a exon I.1 was constructed by inserting a 134-bp SspI-HincII fragment, derived from the Hif1a genomic clone {lambda}H13 (21), into the SmaI site of the pBluescriptSKII vector (Stratagene, La Jolla, CA). The plasmid for Hif1a exon I.2 was constructed by inserting a 218-bp NcoI-StuI fragment, derived from a {lambda}ZAP mouse Hif1a genomic clone, into the SmaI site of the pBluescriptSKII vector. Single-stranded antisense or sense cRNA probes were generated by in vitro transcription of these plasmids using 100 µCi 35S-UTP and T7 or SP6 RNA polymerases as described by the manufacturer (Stratagene). Adult male C57BL/6 mice from an in-house breeding facility were killed by decapitation, both testes were removed, embedded in Tissue Tek O.C.T. (Miles Scientific, Naperville, IL) and transferred into a mixture of methyl butane and dry ice until frozen. The blocks were stored at -70 C. Ten-micrometer sections were cut with a cryostat and melted on silane-coated glass slides. Sections were incubated in 2x SSC (20x SSC = 3 M NaCl, 0.3 M Na3-citrate) at 70 C, digested with Pronase (40 µg/ml), fixed in 4% paraformaldehyde, and acetylated with acetic anhydride diluted 1:400 in 0.1 M triethanolamine. Hybridization was performed in buffer containing 50% formamide, 10% dextran sulfate, 10 mM Tris-HCl (pH 7.5), 10 mM sodium phosphate (pH 6.8), 2x SSC, 5 mM EDTA, 150 µg/ml yeast tRNA, 0.1 mM UTP, 1 mM adenosine 5'-O-(2-thiodiphosphate), 1 mM adenosine 5'-O-(3-thiotriphosphate), 10 mM dithiothreitol, 10 mM 2-mercaptoethanol, and 2.5 x 104 cpm/ml 35S-labeled RNA probe overnight at either 48 C (exon I.1) or 60 C (exon I.2). Slides were washed in 2x SSC/50% formamide at 37 C (exon I.1) or 60 C (exon I.2) for 4 h, digested with RNase (20 µg/ml) for 15 min, washed again with 2x SSC/50% formamide overnight, and dehydrated in graded ethanol. Slides were coated with Kodak NTB-2 emulsion (Eastman Kodak Co., Rochester, NY) diluted 1:1 in water, and exposed for 21 d (exon I.1) or 50 d (exon I.2), respectively. Slides were developed and counterstained with 0.02% Toluidin Blue, air dried, and mounted.

Cell Culture
The following cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA): human HeLa cervic carcinoma (ATCC no. CCL-2); mouse TM3 Leydig cells (CRL-1714); TM4 Sertoli cells (CRL-1715); GC-1 spg spermatogonia (CR-2053); and GC-2 spd(ts) spermatocyte (CRL-2196). The human MCF-7 adenocarcinoma cell line was kindly provided by J. Lisztwan and W. Krek (Basel, Switzerland). The human Jurkat T cell leukemia line was kindly provided by M. Balzer (Zürich, Switzerland). All cell lines were cultured in DMEM (high glucose, Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% heat-inactivated FCS (Roche Molecular Biochemicals, Mannheim, Germany), 100 U/ml penicillin, 100 µg/ml streptomycin, 1x nonessential amino acids, and 1 mM Na-pyruvate (all purchased from Life Technologies, Inc.) in a humidified atmosphere containing 5% CO2 at 37 C. Oxygen partial pressures in the hypoxic workstation (InVivoO2-400, Ruskinn Technology, Leeds, UK) or in the incubator (Forma Scientific, model 3319, Marietta, OH) were either 140 mm Hg (20% O2 vol/vol, normoxia) or 7 mm Hg (1% O2 vol/vol, hypoxia).

Immunoblot Analysis
Nuclear extracts were prepared as described previously (25). Protein concentrations were determined by the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA) or the BCA assay (Pierce Chemical Co., Rockford, IL) using BSA as a standard. Nuclear extracts (50 µg) were electrophoresed through SDS-polyacrylamide gels and elctrotransferred to nitrocellulose membranes (Schleicher & Schuell, Inc., Dassel, Germany) using standard procedures. Membranes were stained with Ponceau S (Sigma, St. Louis, MO) to confirm equal protein loading and transfer. HIF-1{alpha} was detected using an affinity-purified chicken polyclonal IgY antibody raised against a bacterially expressed GST-HIF-1{alpha}530–825 fusion protein (28) followed by a rabbit antichicken secondary antibody (Promega Corp., Madison, WI). ARNT was detected using the mouse monoclonal antibody MA1–515 (Affinity BioReagents, Inc., Golden, CO) followed by goat antimouse secondary antibody (Pierce Chemical Co.). Sp1 was detected using the rabbit polyclonal antibody sc59 (Santa Cruz Biotechnology, Inc., Glaser AG, Basel, Switzerland) followed by a goat antirabbit secondary antibody (Sigma). The horseradish peroxidase-coupled secondary antibodies were detected by using luminol (Sigma) chemiluminescent substrate, exposure to x-ray films (Fuji Photo Film Co., Ltd., Tokyo, Japan) and laser densitometric scanning (Molecular Dynamics Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

Isoform-Specific RT-PCR Analysis
Total RNA was isolated from normoxic and hypoxic cell cultures according to the method described by Chomczynski and Sacchi (36). For cDNA synthesis, 6 µg RNA was heat denatured (3 min at 70 C) and reverse transcribed in 100 µl 50 mM Tris/Cl pH 8.3, 60 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol, 0.5 mM deoxy-NTPs, and 1 U/µl RNAsin (Promega Corp.), using 5 µg (deoxythymidine)12-18 primers (Amersham Pharmacia Biotech) and 250 U Stratascript reverse transcriptase (RT) (Stratagene). After incubation for 30 min at 37 C and for 30 min at 42 C, the reaction was stopped by heating to 95 C for 5 min. An aliquot (2 µl) of each cDNA reaction was subjected to PCR amplification using each 50 pmol of the forward primers mHIFexI.1 (5'-TTTCTGGGCAAACTGTTA-3') and mHIFexI.2 (5'-CGCCTCTGGACTTGTCT-3'), and the reverse primer mHIFexIII (5'-TAACCCCATGTATTTGTTC-3') in 50 µl 1x PCR buffer (Stehelin, Basel, Switzerland), 0.2 mM deoxy-NTPs, and 0.2 U SuperTaq DNA polymerase (Stehelin). After 35 cycles of 94 C for 30 sec, 48 C for 30 sec, and 72 C for 2 min, the PCR products were analyzed by agarose gel electrophoresis and ethidium bromide staining.

Reporter Gene Assays
Two fragments of 1.0 kb and 1.4 kb, corresponding to the exon I.1-specific (21) and the exon I.2-specific promoters (22), respectively, were inserted in front of a luciferase reporter gene. For transient transfections, 0.5 x 106 cells in 350 µl medium without FCS were mixed with 50 µg DNA in 50 µl 10 mM Tris/Cl (pH 7.4), 1 mM EDTA and electroporated at 250 V and 960 microfarads (GenePulser, Bio-Rad Laboratories, Inc.). Luciferase reporter genes were coelectroporated into cells together with the ß-galactosidase reference vector pCMVlacZ (37). The cells were split and incubated for 38–72 h under normoxic or hypoxic conditions. After stimulation, transiently transfected cells were lysed in reporter lysis buffer (Promega Corp.) and luciferase and ß-galactosidase activities were determined according to the manufacturer’s instructions (Promega Corp.) using a Lumat LB9501 luminometer (EG&G Berthold, Bad Wildbad, Germany) and a DigiScan 96-well plate photometer (ASYS), respectively. Differences in the transfection efficiency and extract preparation were corrected by normalization to the corresponding ß-galactosidase activities, and the results were displayed as fold induction above the activity of a promoterless vector.

Immunohistochemistry
For hypobaric exposure, male C57Bl/6C3F1 mice (5–7 months old) were housed in an airtight chamber in which the air pressure was slowly (1 h) lowered to a final pressure of 228 mm Hg (corresponds to a normobaric oxygen concentration of 6%), and kept under these conditions for 6 h. Immediately thereafter, the mice were killed by cervical dislocation, and testis and epididymis were excised and frozen in liquid nitrogen/isopentane. Frozen testes and epididymes of normoxic and hypoxic mice were cut into serial sections of 6 and 2 µm, respectively, dried on a 50 C hot plate for 2 min, and fixed in 4% formaldehyde in PBS for 10 min. All antibodies were diluted in 50 mM Tris/Cl, pH 7.4, 154 mM NaCl, 0.1% Tween 20, 10% FCS. Sections were incubated with the chicken anti-HIF-1{alpha} polyclonal antibody described above or 10% FCS alone overnight at 4 C. A peroxidase-conjugated rabbit antichicken IgY (Pierce Chemical Co.) was incubated for 45 min at RT, followed by a peroxidase conjugated goat antirabbit Ig (DAKO Diagnostika GmbH, Hamburg, Germany) for 45 min. The slices were developed with diaminobenzidine (Sigma) for 15 min and mounted in distrene/dibutyl phthalate/xylene medium.

Immunofluorescence
Sperm cells were obtained from mouse epididymis and vas deferens, washed in PBS and resuspended in DMEM. After incubation under 20% or 1% oxygen for 4 h at 37 C, the sperm cells were fixed with 3.5% formaldehyde, pelleted, resuspended in 80% ethanol, spread on glass slides, and air- dried. After rehydration, unspecific binding sites were blocked with PBS containing 10% FCS. Subsequently, the sperm cells were incubated with the polyclonal anti-HIF-1{alpha} chicken IgY antibody described above or a monoclonal IgG2b antibody (clone H1{alpha}67, Novus Biologicals, Littleton, CO) or 3% BSA in PBS alone. FITC-labeled rabbit antichicken IgY (Promega Corp.) or rabbit antimouse IgG (DAKO Corp.) secondary antibodies were used for the detection by fluorescence microscopy (Axioplan 2000, Carl Zeiss Vision GmbH, Mannheim, Germany).


    ACKNOWLEDGMENTS
 
The authors wish to thank A. Damert; J. Lisztwan, W. Krek, and M. Balzer for the gift of materials; A.-K. Schick for excellent technical assistance; G. Fletschinger for the artwork; P. Attermeyer for technical support with microscopy; and D. M. Stroka and M. Bergmann for helpful advice.


    FOOTNOTES
 
This work was supported by grants from the Max-Planck-Society (to H.H.M.), the Medical University of Lübeck (to D.M.K., K.F.W. and R.H.W.), and the Deutsche Forschungsgemeinschaft (We2672/1-1, to R.H.W.).

H.H.M. and D.M.K. contributed equally to this work.

1 Present address: Institute of Physiology, University of Zürich-Irchel, CH-8057 Zürich, Switzerland. Back

Abbreviations: HIF, Hypoxia-inducible factor; TBP, TATA-binding protein.

Received for publication July 10, 2001. Accepted for publication November 1, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 Discussion
 MATERIALS AND METHODS
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