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Male Reproductive Tract |
Department of Biology,3 Monmouth University, West Long Branch, New Jersey 07764
Department of Anatomy and Cell Biology,4 McGill University, Montreal, Quebec, Canada H3A 2B2
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß subunits, HIF-1 activates genes that mediate oxygen homeostasis and cell survival pathways or trigger cell death responses. Previously we have shown that HIF-1 mRNA is expressed in the adult rat epididymis. Goals of this study were to determine whether HIF-1 protein is activated by ischemia in the rat epididymis, to determine whether epididymal HIF-1 mRNA expression is androgen dependent, and to identify epididymal cell types expressing HIF-1 and ß. Immunoblot analysis revealed that HIF-1 protein is primarily present in corpus and cauda of the normoxic epididymis and unaffected by ischemia, whereas HIF-1ß was detected equally in all regions and also unaffected by ischemia. HIF-1 mRNA expression in all regions was not affected by 15 days bilateral orchiectomy. Principal cells stained positive for HIF-1 by immunocytochemistry, with the epithelium of initial segment and caput epididymidis staining less intensely than corpus and cauda. HIF-1ß immunoreactivity was equally present in principal cells in all regions. Clear, narrow, and basal cells were unreactive for HIF-1 and ß. The presence of HIF-1 in normoxic epididymis and the regional distribution of HIF-1 suggests fundamental differences in how proximal and distal regions of the epididymis maintain oxygen homeostasis to protect the epithelium and spermatozoa from hypoxia.
epididymis, gene regulation, male reproductive tract, sperm, stress
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The ability to maintain oxygen homeostasis is essential to the survival of all aerobic organisms. Mammalian cells are extremely sensitive to changes in oxygen concentration, particularly decreases in local oxygen supply (hypoxia). Organisms rely on a number of oxygen-sensing mechanisms to detect hypoxia and stimulate molecular adaptations to respond accordingly [3–5]. When subjected to hypoxia, most cells rapidly increase transcription of specific genes involved in oxygen homeostasis in an effort to cope with hypoxic conditions [6, 7].
Hypoxia-inducible factor-1 (HIF-1) is considered the master regulator of the response to hypoxia [3, 8]. HIF-1 is a basic helix-loop-helix transcription factor that helps to restore oxygen homeostasis by activating a variety of hypoxia-sensitive genes involved in cellular processes such as angiogenesis, erythropoiesis, glycolysis, and apoptotic and proliferative responses to ischemia and hypoxia [9]. HIF-1 can activate both cell survival and cell death genes, depending on the extent and duration of oxygen debt [5, 10]. Many investigators are studying functions of HIF-1 during embryogenesis, tumor vascularization and progression, and ischemic processes of clinical importance [5, 11].
Active HIF-1 is a heterodimer consisting of one and one ß subunit. HIF-1 subunits are widely expressed in mammalian tissues from virtually all organisms studied to date [5, 7]. HIF-1 is activated by hypoxia. Under normoxic conditions, the von Hippel-Lindau tumor suppressor protein targets HIF-1 subunits for polyubiquitination and degradation in the proteasome [12]. The HIF-1ß subunit, also called ARNT (arylhydrocarbon nuclear translocator), is constitutively produced and unregulated by oxygen tension [13]. When intracellular oxygen reaches a critically low threshold, HIF-1 subunits are rapidly protected from proteasomal degradation, allowing HIF-1 and HIF-1ß subunits to associate and form active HIF-1.
Metabolic activity of the epididymis critically depends on the continuous delivery of nutrients and oxygen to the epididymal epithelium via an elaborate microvasculature that varies in complexity along the duct [14]. Maintaining a properly oxygenated microenvironment is essential for epididymal physiology and for protecting maturing spermatozoa from oxidative and hypoxic damage [1, 2, 15]. We hypothesized that HIF-1 plays a key role in mediating oxygen homeostasis in the epididymis [16]. Previously we demonstrated that HIF-1 mRNA is expressed in the adult rat testis and epididymis. The purpose of this study was to test the hypothesis that HIF-1 protein is activated by ischemia in the rat epididymis, to determine whether epididymal HIF-1 mRNA expression is androgen dependent, and to determine the epididymal cell types producing HIF-1 and HIF-1ß subunits.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Adult male Sprague-Dawley rats (450–600 g) were purchased from Charles River Laboratories (Stoneridge, NY). Animals were housed two per cage at Monmouth University under controlled light (12L:12D) and temperature with free access to food and water. All aspects of animal handling and surgery were conducted in accordance with appropriate animal welfare criteria established by the National Research Council (NCR) publication Guide for Care and Use of Laboratory Animals (copyright 1996, National Academy of Science).
Surgical Manipulations
Animals were anesthetized with halothane (Sigma-Aldrich, St. Louis, MO), and surgeries performed via a midline laparotomy using sterile procedures. To determine whether HIF-1 subunits are activated by ischemia and hypoxia, unilateral experimental ischemia was created by placing a ligature of 4.0 silk suture around internal spermatic artery and vas deferential artery for 1 h. These surgical conditions are known to render the testis and epididymis ischemic and hypoxic [17]. The testis and epididymis were returned to the scrotum and the laparotomy incision was clamped closed. Studies were also carried out with normoxic, untreated tissues and contralateral tissues were used as sham-operated controls [16]. Rats were maintained under anesthesia for the duration of the surgical treatment prior to being killed. Rats (n = 5) were killed with carbon dioxide and their epididymides dissected free of adipose tissue, cut into four regions (initial segment, caput, corpus, and cauda), and immediately frozen in liquid nitrogen and stored at -70°C prior to nuclear protein extraction and RNA isolation. Ischemic kidneys were used as control tissues to detect HIF-1 protein. Kidney ischemia was created by placing a ligature around renal artery for 1 h prior to removing the organ.
For androgen regulation studies, animals were anesthetized with halothane and orchiectomy surgeries performed via a midline laparotomy as previously described [18]. Nine rats were divided into three groups of three animals per group (n = 3) as follows: 1) bilaterally sham-orchiectomized controls simultaneously implanted s.c. with an empty 3.5-cm polydimethylsiloxane capsule (Silastic medical grade tubing; Dow-Corning, Midland, MI), 2) bilateral orchiectomized (orch) rats implanted with an empty 3.5-cm capsule, and 3) bilateral orchiectomized rats implanted with a 3.5-cm capsule filled with testosterone (Sigma-Aldrich). Capsules were prepared and presoaked for 48 h in 1x PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4 [pH 7.3]) containing 4% BSA according to the procedure of Berndtson et al. [19] as described previously [20]. Following orchiectomy, the epididymis was returned to the scrotum and abdominal muscle and skin sutured closed. Animals were killed with carbon dioxide 15 days after surgery, and their epididymides trimmed free of fat and dissected into four regions (initial segment, caput, corpus, and cauda). Tissues, including the kidney, were immediately frozen in liquid nitrogen and stored at -70°C prior to RNA isolation.
Chemiluminescence immunoassays of serum testosterone were carried out at the University of Virginia Health System General Clinical Research Center Core Laboratory, Charlottesville, VA, using a Bayer ACS180 system (Bayer Diagnostics, Tarrytown, NJ). Inter- and intraassay coefficients of variations were 6.82% and 8.68%, respectively. Seminal vesicle and prostate wet weights were also determined to assess the biological activity of T concentrations in orch rats receiving T implants.
RNA Isolation and RT-PCR Analysis of mRNA Expression
Total RNA was isolated from frozen tissues using TRIReagent according to the manufacturer's instructions (Molecular Research Center Inc., Cincinnati, OH). HIF-1 forward 5'-TGCTTGGTGCTGATTTGTGA-3' (nt 681–700) and reverse primers 5'-GGTCAGATGATCAGAGTCCA-3' (nt 871–890) were designed from the rat HIF-1 cDNA sequence (AF057308) [21] using JellyFish software (version 1.1; LabVelocity, Inc., Burlingame, CA) and synthesized by MWG-Biotech (High Point, NC). These primers amplify a 209-bp fragment of the rat HIF-1 gene.
HIF-1 was coamplified by multiplex relative reverse transcription-polymerase chain reaction (RT-PCR) analysis with mouse ß-actin (Stratagene, La Jolla, CA) primers as internal controls. Primer concentrations were optimized to amplify ß-actin PCR products of 514 bp in the same linear range as HIF-1 amplicons. Amplifications were carried out using a Techne Genius thermal cycler (Techne Inc., Burlington, NJ). One microgram of total RNA was reverse transcribed and amplified by the one-step AccessQuick RT-PCR procedure (Promega Corp., Madison, WI) in a 50-µl reaction volume containing 50 pmol of each HIF-1 primer and 6.25 pmol of each ß-actin primer in the presence of AMV reverse transcriptase and Tfl DNA polymerase. Reverse transcription was carried out at 48°C for 45 min, followed by denaturation at 94°C for 2 min and 40 cycles of PCR with a denaturing step at 94°C for 30 sec, an annealing step at 60°C for 1 min, an elongation step at 68°C for 2 min, and a final extension at 68°C for 7 min. Eight-microliter aliquots of PCR products were electrophoresed through 2% agarose gels in 1x Tris-borate-EDTA buffer. Gels were stained with ethidium bromide and images captured with a ChemiDoc gel documentation system (Bio-Rad Laboratories, Hercules, CA). RT-PCR controls routinely analyzed included a single primer pair-positive control amplification, no primer, and no reverse transcriptase negative controls. All RT-PCR products were verified by Southern blot analysis as previously described [16] and by DNA sequencing of products cloned into pGEM-T Easy plasmid vectors (Promega Corp.).
To evaluate angiotensinogen mRNA expression as a measure of ischemia and hypoxia, RT-PCR was carried out using forward (5'-TTCAGGCCAAGACCTCCC-3') and reverse primers (5'-CCAGCCGGGAGGTGCAGT-3'), which amplify a 312-bp fragment (nt 245–557) of the rat angiotensinogen gene (NM_134432) [22].
Nuclear Protein Extraction
Frozen tissues were pulverized in liquid nitrogen using a ceramic mortar and pestle, and nuclear and cytoplasmic protein extracts were prepared using NE-PER extraction reagents according to the manufacturer's instructions (Pierce, Rockford, IL). Extraction solutions contained a cocktail of the following protease inhibitors and reducing agents: aprotinin (2 µg/ml), dithiothreitol (0.5 mM), leupeptin (2 µg/ml), pepstatin (2 µg/ml), and phenylmethylsulfonyl fluoride (2 mM). Protein concentrations were determined by the Bradford assay (Bio-Rad Laboratories) using BSA as the standard.
Isolation of Epididymal Spermatozoa and Extractionof Sperm Proteins
Epididymides with a small proximal portion of their vasa deferentia attached were trimmed free of adipose tissue and dissected into initial segment, caput, corpus, and cauda regions. Each region was immersed in ice-cold 1x PBS on ice. Initial segment, caput, and corpus regions were sliced several times with a razor blade and spermatozoa released into PBS for 20 min. Spermatozoa from cauda epididymidis were isolated by making an incision in distal cauda, inserting a 28-gauge needle into the vas deferens and back-flushing cauda tubules with glycerol containing blue food coloring dye to release spermatozoa into PBS. Epididymal spermatozoa were pelleted by centrifugation at 800 x g for 5 min at 4°C. Sperm concentration was estimated by hemocytometric counting. Preparations contained less than 5% somatic cell contamination. Nuclear proteins were isolated from approximately one million freshly isolated epididymal spermatozoa from each region using Pierce NE-PER extraction reagents according to the manufacturer's instructions (Pierce). Protein isolations were carried out on single pieces of tissue from each epididymal region. Experiments were repeated with tissue from three different animals (n = 3).
Immunoblot Analysis
Nuclear proteins were separated by denaturing SDS-PAGE through 10% polyacrylamide gels (Bio-Rad Laboratories) according to the method of Laemmli [23]. HIF-1 and HIF-1ß/ARNT fusion proteins (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) were included as positive controls for antibody specificity. Proteins were transferred to NitroBind nitrocellulose (Osmonics Inc., Westborough, MA) by electroblotting and blots were stained with Ponceau S (0.005% in 1% acetic acid) to confirm that equal amounts of protein were electrophoresed and transferred. Blots were blocked in 1x western wash (50 mM Tris, 30 mM NaCl, 0.001% Tween 20 [pH 7.6]) containing 5% nonfat dry milk for 30 min to 2 h at room temperature with gentle agitation. To detect HIF-1, blots were incubated overnight at 4°C in 1x western wash, 5% nonfat dry milk containing a 1:500 dilution of mouse monoclonal antibody (NB100-105; Novus Biologicals Inc., Littleton, CO). Blots were stripped of bound antibody by incubating in Restore Western blot stripping buffer (Pierce) for 15 min at room temperature, washed in 1x western wash for 5 min, and then blocked prior to subsequent immunoprobing for HIF-1ß or -tubulin.
HIF-1ß was detected by immunoprobing blots with a 1:2000 dilution of rabbit anti-HIF-1ß/ARNT polyclonal antibody (NB 100-110, Novus Biologicals Inc.). Alpha tubulin was detected on immunoblots as a loading control for protein quantitation using a 1:2000 dilution of mouse anti--tubulin monoclonal antibody (T-5168; Sigma-Aldrich). Blots were washed three times in 1x western wash for 35 min and incubated for 2 h in 1x western wash, 5% nonfat dry milk containing a 1:10 000 dilution of goat anti-mouse IgG (SC2005; Santa Cruz Biotechnology) conjugated to horseradish peroxidase (HRP) to detect HIF-1 and -tubulin or goat anti-rabbit IgG-HRP (SC2004) to detect HIF-1ß. Blots were washed extensively in three changes of 1x western wash for 35 min and then in 1x western wash, 1% nonfat dry milk for 10 min. Blots were developed by enhanced chemiluminescence using a Pierce SuperSignal West Pico kit and exposed to x-ray film (BioMax ML, Kodak, Rochester, NY). To demonstrate HIF-1 and HIF-1ß antibody specificity, all blots were stripped and reprobed with primary antibody blocked by preincubation with a 5-fold excess of blocking peptide (HIF-1 peptide, sc-8711P; HIF-1ß peptide, sc8077P; Santa Cruz Biotechnology) for 2 h at 4°C prior to overnight incubation with blots followed by detection with secondary antibodies as described above.
Immunocytochemistry
Adult rats were anesthetized with an i.p. injection of sodium pentobarbital (Somnitol, MTC Pharmaceuticals, Hamilton, ON, Canada) and perfused through the abdominal aorta with Bouin fixative. Epididymides were trimmed free of adipose tissue and dissected into initial segment, caput, corpus, and cauda regions and then sliced longitudinally and immersed in Bouin fixative overnight. Following fixation, tissues were dehydrated in three changes of 70% ethanol and eventually dehydrated in a series of graded ethanol solutions, followed by dioxane and embedding in paraffin wax. Sections (5 µm thick) were cut, mounted on glass slides, and processed for light-microscopic immunocytochemical analysis.
The following affinity-purified antibodies were used at the various dilutions for routine peroxidase immunostaining: polyclonal goat anti-human HIF-1 (1:150; Santa Cruz Biotechnology), monoclonal mouse anti-human HIF-1 (1:100; Novus Biologicals), and polyclonal rabbit anti-human HIF-1ß (1:150; Novus Biologicals). Negative control experiments were performed on adjacent sections by substituting 1x Tris-buffered saline (TBS) buffer for the primary antibody.
The paraffin sections were deparaffinized with Histoclear (AGTC Bioproducts, Ltd., Huntingdon, UK) and hydrated in a series of graded ethanol solutions. Endogenous peroxidase activity was inactivated with 70% ethanol containing 1% hydrogen peroxide, whereas residual picric acid was neutralized in 70% alcohol containing 1% lithium carbonate. After hydration, the tissues were washed in distilled water containing 300 mM glycine to block free-aldehyde groups. Prior to immunostaining, the sections were blocked for 15 min with 10% goat serum in TBS. Tissue sections were incubated at 37°C in a humidified chamber for 90 min with 100 µl of diluted primary antibody. Following several washes in TBS containing 0.1% Tween-20, the sections were blocked with 10% goat serum for 15 min to prevent nonspecific binding of the secondary antibody. The secondary antibody incubation was performed at 37°C with anti-rabbit IgG conjugated to horseradish peroxidase at a dilution of 1:250 with TBS. All sections were washed and incubated with peroxidase substrate: 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.03% hydrogen peroxide in TBS. The sections were counterstained with 0.1% methylene blue and dehydrated in a graded series of ethanol solution and Histoclear. Cover slips were mounted onto glass slides with Permount (Fisher Scientific, Pittsburgh, PA).
Quantitation of Results and Statistical Analysis
RT-PCR gel images were captured using a ChemiDoc gel documentation system and quantitation of results carried out with Quantity One quantitation software (version 4; Bio-Rad Laboratories). Integrated peak areas for HIF-1 and angiotensinogen PCR products were normalized to integrated peak areas for ß-actin PCR products to control for variations in intersample amplification and gel loading. HIF-1 and HIF-1ß protein levels were normalized to -tubulin as a loading control. Data were analyzed by one-way ANOVA and results considered significantly different at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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mRNA in the Ischemic Epididymis
Previously we demonstrated that HIF-1 mRNA is equally expressed in all regions of the normoxic adult rat epididymis [16]. We also examined HIF-1 mRNA expression during postnatal development of the rat epididymis and showed that HIF-1 mRNA is present at the time of birth (Postnatal Day 0) and expressed at relatively equal abundance through adulthood (data not shown). To determine whether epididymal HIF-1 mRNA expression is regulated by ischemia, total RNA from 1 h ischemic epididymides was analyzed by RT-PCR analysis. Expression of HIF-1 mRNA in all regions of the epididymis was unaffected by 1 h of ischemia, compared with normoxic and sham-operated controls (Fig. 1).
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To confirm that our surgical treatment was creating ischemia in the epididymis, RNA from normoxic and 1 h ischemic corpus epididymidis was analyzed for angiotensinogen (AGT) mRNA expression by RT-PCR (Fig. 2A). AGT mRNA expression has been shown to be hypoxia dependent following chronic ischemia and hypoxia in the rat epididymis [22]. AGT mRNA expression in ischemic corpus epididymidis was significantly reduced (P < 0.05), compared with the normoxic corpus epididymidis (Fig. 2B).
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HIF-1 and HIF-1ß Protein in the Epididymis
To determine whether HIF-1 and HIF-1ß protein subunits were produced in the adult rat epididymis and activated by ischemia, we isolated nuclear proteins from the initial segment, caput, corpus, and cauda epididymidis of both normoxic and 1 h ischemic epididymides and carried out immunoblot analysis with antibodies to each subunit. HIF-1 was abundant in nuclear protein extracts from the normoxic epididymis and immunoblot analysis revealed regional differences in the abundance of HIF-1 protein (Fig. 3A). HIF-1 polypeptide with a relative migration of approximately 90 kDa was primarily detected in corpus and cauda epididymidis (Fig. 3A). HIF-1 was faintly detected in initial segment and caput epididymidis after prolonged exposure of immunoblots. For HIF-1ß a 93-kDa polypeptide was detected in all regions of the epididymis (Fig. 3B).
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Quantitation of HIF-1 mRNA and protein data revealed no statistically significant differences (P < 0.05) in the relative abundance of steady-state HIF-1 protein and mRNA in any regions of the epididymis following 1 h of ischemia (Fig. 4). Surprisingly, the relative abundance of HIF-1 protein was not significantly affected by 1 h of ischemia in any regions of the epididymis (Fig. 4). Similar results were obtained with cytoplasmic protein extracts (data not shown). Experiments were also carried out on epididymides after 15 min and 6 h of ischemia, with the results being identical to the 1 h treatment (data not shown). As expected, HIF-1ß protein was unaffected by 1 h of ischemia (Fig. 4). The specificity of the commercially available antibodies for HIF-1 and HIF-1ß used in these experiments was determined by preincubating each antibody with blocking peptide prior to incubation with immunoblots. No specific bands of the expected sizes were detected for HIF-1 or HIF-1ß (Fig. 5).
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Androgens Do Not Influence Epididymal HIF-1 mRNA Expression
Androgen regulation studies were carried out to determine whether HIF-1 mRNA expression in the epididymis is dependent on circulating androgens. Peripheral blood serum testosterone concentrations (mean SEM; ng/ml) in rats after 15 days of sham orchiectomy, bilateral orchiectomy (orch), and orchiectomy with testosterone replacement (orch + T) were published previously [18] and are as follows: sham, 0.99 ± 0.12; 15 days orch, 0.17 ± 0.08; and 15 days orch + T, 1.41 ± 0.09. Serum T concentrations were significantly repressed in orch rats, compared with those in sham control rats (P < 0.05). Serum T concentrations in orch + T rats were elevated, compared with those in sham rats and orch rats at 15 days (P < 0.05). Expression of HIF-1 mRNA in all regions of the epididymis was unaffected after 15 days of orch or 15 days of orch + T (Fig. 6). These studies indicate that expression of HIF-1 mRNA is not androgen dependent in the adult rat epididymis.
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Immunoblot Analysis of HIF-1 Protein in Epididymal Spermatozoa
It has been reported that a testis-specific isoform of HIF-1 is present in the midpiece of mouse spermatozoa [24]. To determine whether HIF-1 subunits are present in rat spermatozoa, nuclear proteins from epididymal spermatozoa were examined by immunoblot analysis. HIF-1 and HIF-1ß were not detected in epididymal spermatozoa from 1 h ischemic epididymides (Fig. 7) or normoxic epididymides (data not shown). Testicular spermatozoa from 1 h ischemic testes showed a faint signal for HIF-1 and strong signal for HIF-1ß(Fig. 7).
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Immunocytochemical Localization of HIF-1 and HIF-1ß
To localize HIF-1 to specific cells types in the epididymis, immunocytochemical studies were carried out on normoxic and 1 h ischemic sections of adult rat epididymis. In the epididymis of normoxic animals, a cell type- and region-specific pattern of staining was noted with the anti-HIF-1 antibody. Principal cells of the corpus and cauda regions were intensely reactive in contrast to the weak staining noted for these cells in the initial segment and caput epididymidis (Fig. 8). Immunostaining in the corpus and cauda epididymidis also revealed that the cytoplasm of many principal cells were intensely reactive, as were their nuclei (Fig. 8). However, while some principal cells showed a strong cytoplasmic signal, their nucleus appeared weakly reactive or unreactive. Throughout the epididymis, clear, narrow, and basal cells were consistently unreactive for anti-HIF-1 antibody (Fig. 8).
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Throughout the epididymis, principal cells were also reactive for HIF-1ß. However, with the exception of the initial segment, which revealed slightly weaker levels of reaction product, relatively equal levels of moderate reactivity were detected for these cells in the caput, corpus, and cauda regions (Fig. 9). In the latter regions, many nuclei of principal cells were as reactive as their cytoplasm (Fig. 9). As was observed for HIF-1, clear, narrow, and basal cells were unreactive for HIF-1ß (Fig. 9). A qualitative assessment of the localization of HIF-1 and HIF-ß in the epididymis is shown in Table 1. No differences in the cellular localization of either HIF-1 or HIF-1ß were apparent when normoxic and ischemic sections were analyzed. Immunostaining for HIF-1 subunits was not observed in control sections incubated without primary antibody (inset, Fig. 8a).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and HIF-1ß protein subunits are present in the epididymis and hypothesize that HIF-1 is important for maintaining oxygen homeostasis in the epididymis. As expected, HIF-1ß was constitutively present in all regions of both the normoxic and ischemic epididymis. This was an expected result because in virtually all tissues studied to date, HIF-1 protein is an oxygen-dependent subunit that is rapidly stimulated by decreasing concentrations of oxygen, whereas HIF-1ß is a constitutively expressed protein [3, 8].
However, an unexpected finding was that HIF-1 is highly localized in the normoxic epididymis in a region-specific manner and unregulated by ischemia. By both immunoblot analysis and immunocytochemistry, HIF-1 was primarily detected in the corpus and cauda, and only very faintly present in initial segment and caput epididymidis. It has been well established that the hypoxic upregulation of HIF-1 protein and down-regulation under normoxia is controlled through changes in protein stability [3, 8]. The von Hippel-Lindau tumor suppressor protein binds to the protein stabilization domain of HIF-1 to assemble a complex with E3 ubiquitin ligase, which targets HIF for polyubiquitination and subsequent proteosomal degradation [25, 26]. For epididymal HIF-1, although we have not directly measured rates of HIF-1 translation or degradation, it appears that the amount of steady-state HIF-1 in the nucleus is the same under normoxic and hypoxic conditions, suggesting that there is no difference in the amount of HIF-1 entering the nucleus under ischemic conditions.
The significance of having HIF-1, and presumably active HIF-1, in the normoxic epididymis is not known. We propose that the regional distribution of HIF-1 and its abundance in the normoxic epididymis may be directly related to the oxygenation status of the epididymal luminal microenvironment for several reasons. It has been suggested that spermatozoa encounter a progressively more oxygenated luminal microenvironment as they move from the testis into distal regions of the epididymis [27]. In addition, spermatozoa produce ROS, and spermatozoa are highly susceptible to oxidative damage by ROS [15]. Several proteins involved in protection against oxidative stress are abundant in the corpus and cauda including superoxide dismutase [28] and enzymes of the glutathione antioxidant system such as the glutathione S-transferases [29], glutathione peroxidase [30], and 
-glutamyl transpeptidase [20]. The presence of ROS in the luminal fluid and spermatozoa may be relevant to the activation of HIF-1 in the normoxic epididymis because several in vitro studies on HIF-1 biochemistry have provided evidence that HIF-1 may be stabilized through mechanisms that involve oxidative modifications by ROS [31]. It is possible that ROS stabilization of HIF-1 activates HIF-1 to provide the epididymal epithelium with protection against ROS, thus indirectly contributing to the protection of spermatozoa in the lumen.
Alternatively, perhaps local hypoxic microenvironments exist in the epididymis. The capillary networks of distal regions of the epididymis are less elaborate than in proximal regions, particularly the initial segment [32], and blood flow in distal regions is lower than in proximal regions [14]. In addition, tubule diameter in the cauda is the largest of all epididymal regions creating a large diffusion distance for movement of oxygen into the lumen. Together, these anatomical features may create small local fluctuations in oxygen tension in distal regions. Consistent with this idea, oxygen consumption by the large number of spermatozoa stored in distal regions of the epididymis may necessitate additional protection against fluctuations in oxygen tension. It has also been proposed that low oxygen tensions may exist within densely packed populations of spermatozoa in cauda epididymidis [15]. Such areas of localized hypoxia may in fact be another reason for activation of HIF-1 in the corpus and cauda. We are presently involved in studies to evaluate these aspects of oxygen homeostasis and the potential involvement of HIF-1.
Another explanation is that ubiquitination and degradation pathways for HIF-1 in the normoxic epididymis are different from the pathways characterized in other tissues. Regardless of the mechanisms stabilizing HIF-1, its abundance in corpus and cauda, compared with initial segment and caput, suggests fundamentally different processes for HIF-1-mediated oxygen homeostasis in proximal and distal regions of the epididymis.
It is well known that a variety of different genes in the epididymis are expressed in an androgen-dependent manner [33–36], and thus we were interested in determining whether HIF-1 is androgen regulated. Our results, following 15 days of orch, a time point at which circulating and luminal concentrations of T are virtually depleted, or orch + T showed that HIF-1 mRNA expression is not affected by androgens.
To complete our understanding of the distribution of HIF-1 in the epididymis, we examined the cellular localization of HIF-1 and HIF-1ß subunits by immunocytochemistry and immunoblot analysis. The immunocytochemical studies confirmed and extended our immunoblotting studies by showing that immunoreactivity for HIF-1 in the epididymal epithelium was stronger in the corpus and cauda epididymidis than in initial segment and caput, whereas HIF-1ß immunoreactivity was relatively equal in all regions of the epididymis. Not surprisingly, HIF-1 and HIF-1ß immunoreactivity was primarily detected in principal cells throughout the epididymis. Clear cells of the epididymis, which are known to have endocytic functions [1], were unreactive for both HIF-1 and HIF-1ß. In a recent study by Marti et al. [24], it was reported that proximal segments of the mouse epididymis were not immunoreactive for HIF-1 and that HIF-1 is present under both normoxic and hypoxic conditions in the midpiece of epididymal spermatozoa [24]. However, this work identified a truncated, testis-specific isoform of HIF-1 (mHIF-1I.1) in epididymal spermatozoa that is unaffected by hypoxia. Mouse epididymal spermatozoa were not immunoreactive for the mouse isoform of HIF-1 (mHIF-1I.2), which is similar to the human and rat forms of HIF-1 [24, 37].
To determine whether HIF-1 is present in rat spermatozoa, we isolated nuclear proteins from testicular and epididymal spermatozoa for immunoblot analysis. Although HIF-1 was faintly detected in preparations of testicular spermatozoa, we believe these results may be due to slight contamination of sperm preparations by parenchymal cells because of our isolation technique of using minced tissue to obtain testicular sperm. The immunoblotting experiments also showed that HIF-1 subunits were not detected in epididymal spermatozoa, and these results were confirmed by immunocytochemistry.
If HIF-1 is not active in epididymal spermatozoa, we hypothesize that HIF-1 has a protective role for the epididymal epithelium, which in turn confers protection of spermatozoa by supporting proper functions of the epididymal epithelium. HIF-1 is known to specifically activate a large number of genes by binding to DNA via the hypoxia response element consensus sequence 5'-RCGTG-3' [3, 38]. HIF-1 target genes are involved in processes such as angiogenesis, erythropoiesis, glucose transport, glycolysis, cell proliferation, and apoptotic and antiapoptotic responses to ischemia and hypoxia [3, 5, 9–11]. To begin to consider potential target genes for HIF-1 in the epididymis, an analysis of upstream promoter sequences for genes known to be expressed in the human, rat, or mouse epididymis that contain the HIF-1 consensus DNA-binding site were identified using Genomatix MatInspector software (version 3.0.03; http://genomatix.de/) and matched to TestisBank (http://medweb.uni-muenster.de/TestisBank/).
This survey revealed several epididymal genes with one or more consensus DNA-binding sites for HIF-1, suggesting the potential regulation of important target genes by epididymal HIF-1. Some noteworthy candidate genes that are expressed in distal regions of the rat and/or mouse epididymis include those involved in apoptotic pathways such as bax [36], bcl-2 [36], and Mcl-1 [36], second-messenger pathways (c-fos [34]), and ROS metabolism (superoxide dismutase; SOD [28]). It is particularly interesting that SOD may be a target for epididymal HIF-1. Consistent with our hypothesis that HIF-1 activation in the corpus and cauda may be a result of ROS, mRNA expression for SOD is highest in rat cauda epididymidis, in which a secreted form of luminal SOD plays a role in protecting sperm from oxidative stress [28]. Other possible targets include genes involved in glucose metabolism (hexokinase [39]) and iron metabolism (transferrin receptor [40]). Also, the erythropoietin gene, which has recently been shown to be induced by hypoxia in the mouse epididymis [41], is a known target of HIF-1 in the kidney.
Another well-characterized HIF-1 target gene is vascular endothelial growth factor (VEGF), which has important roles in angiogenesis and vascular permeability [42, 43]. In the mouse epididymis, VEGF is expressed in distal regions of the duct in which it appears to play a role in vascular permeability and may have both endothelial and nonendothelial target cells [44]. It is also interesting to note that patients with von Hippel-Lindau disease, an autosomal dominant hereditary syndrome caused by germline mutations of the VHL gene, show pathologies characterized by a multitumor phenotype affecting epithelial tissues of the pituitary, pancreas, kidney, and epididymis [45]. Many of these defects are the result of hypervascularization because of VEGF overexpression. In patients with von Hippel-Lindau, the mutant VHL protein does not trigger degradation of HIF-1 under normoxia; thus, HIF-1 is constitutively activated, and through stimulation of target genes such as VEGF, HIF-1 is a causative agent of tumor formation in tissues such as the kidney [5, 46]. The epididymal pathology in patients with von Hippel-Lindau is bilateral epididymal papillary cystadenomas that show elevated expression of VEGF mRNA [42]. Although the expression of HIF-1 in these tumors has not been reported, it is likely that overactivation of HIF-1 may be involved in VEGF overexpression and the epididymal phenotype observed in this disease. Investigating HIF-1 activation of putative target genes such as VEGF will be an important step toward understanding the role of HIF-1 in the epididymis.
In summary, results presented in this study strongly support a role for HIF-1 in oxygen homeostasis in the epididymis. The abundance of HIF-1 in the corpus and cauda epididymidis suggests fundamental differences in how proximal and distal regions of the epididymis regulate oxygen homeostasis through HIF-1-mediated pathways. Additional studies are necessary to further characterize the activation of HIF-1 in the normoxic epididymis and elucidate the role(s) of epididymal HIF-1 in regulating the expression of target genes that may be involved in oxygen homeostasis in the epididymis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: Michael A. Palladino, Biology Department, Monmouth University, 400 Cedar Ave., West Long Branch, NJ 07764. FAX: 732 263 5243; mpalladi{at}monmouth.edu
Received: 11 September 2003.
First decision: 8 October 2003.
Accepted: 1 December 2003.
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) protein in the adult rat testis following ischemic injury occurs without an increase in HIF-1 messenger RNA expression. Biol Reprod 2002 67:995-1002This article has been cited by other articles:
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