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Regular Article |
) Protein in the Adult Rat Testis Following Ischemic Injury Occurs Without an Increase in HIF-1 Messenger RNA Expression1
a Biology Department, Monmouth University, West Long Branch, New Jersey 07764
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and ß subunits. Stabilized from proteasome degradation and activated by hypoxia, HIF-1 stimulates expression of hypoxia-sensitive genes that mediate oxygen homeostasis in many tissues. Our hypothesis is that HIF-1 is involved in the cellular response to hypoxia in the ischemic testis. Goals of this study were to determine if HIF-1 mRNA is expressed in the testis, epididymis, and accessory sex glands of adult Sprague-Dawley rats and to determine if HIF-1 mRNA and protein expression in the testis is affected by experimentally induced ischemia. Total RNA from reproductive organs of adult rats was analyzed by relative reverse transcription-polymerase chain reaction (RT-PCR) analysis. HIF-1 mRNA showed equal expression in testis, all segments of epididymis, ductus deferens, accessory sex glands, and penis. To examine the effects of ischemia on HIF-1 mRNA and protein expression in the testis, rats were subjected to unilateral testicular ischemia by placing a ligature around spermatic artery or ischemia-inducing experimental torsion and reperfusion. RT-PCR revealed that HIF-1 mRNA expression at all times of ischemic treatment and reperfusion was unchanged compared with normoxic controls. HIF-1 protein was detected by immunoblot analysis of nuclear protein extracts from normoxic testes. Steady-state levels of HIF-1 protein were stimulated by 15 min of ischemia and showed a 2-fold increase at 30 min and 1, 3, and 6 h. HIF-1 protein was also elevated by experimental torsion and reperfusion compared with normoxic controls. These results support the hypothesis that HIF-1 may play a role in the cellular response to hypoxia in the ischemic testis.
epididymis, gene regulation, male reproductive tract, stress, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Testicular torsion is a medical emergency and a source of morbidity, especially in neonatal or adolescent males [2, 3]. Rotation of the testis and torsion of the spermatic cord leading to a reduction or blockage in blood flow to the testis can result in testicular ischemic injury, including germ-cell loss, aspermatogenesis, and testicular atrophy [4, 5]. Torsion-induced testicular ischemia often requires surgical interventions such as detorsion or orchiectomy [6].
Cellular and molecular mechanisms responsible for posttorsion tissue damage in the ischemic testis are complex. Some parameters of Sertoli and Leydig cell function appear to be generally maintained in the ischemic testis [7, 8]. It is well documented, however, that torsion leading to testicular ischemia can result in germ-cell death and irreversible aspermatogenesis [4, 9].
Germ-cell-specific apoptosis has been shown to occur following torsion-induced testicular ischemia and ischemia-reperfusion injury to the testis [5, 10, 11]. Leukocyte infiltration and subsequent release of reactive oxygen species (ROS) is thought to contribute to apoptotic pathways responsible for germ-cell death in the ischemic testis [5, 10, 12]; however, it is likely that several mechanisms contribute to cell damage during periods of testicular ischemia.
Mammalian cells are extremely sensitive to changes in oxygen concentration, particularly oxygen debt (hypoxia). Most cells rely on a number of oxygen-sensing mechanisms to detect hypoxia and stimulate molecular adaptations to respond accordingly [13, 14]. When subjected to hypoxia, many cells rapidly increase transcription of a subset of genes involved in oxygen homeostasis in an effort to cope with hypoxic conditions [15, 16].
Hypoxia-inducible factor-1 (HIF-1) is an important regulator of the response to hypoxia and oxygen homeostasis in many tissues [13, 17]. HIF-1 is a member of the basic helix-loop-helix/PER-ARNT-SIM superfamily of transcription factors [18]. HIF-1 activates a variety of target genes involved in cellular processes such as angiogenesis, erythropoiesis, glycolysis, and apoptotic and proliferative responses to ischemia and hypoxia [13, 19, 20].
Active HIF-1 is a heterodimer consisting of one and one ß subunit. HIF subunits are widely expressed in mammalian tissues from virtually all organisms studied to date [21, 22]. Active HIF-1 is primarily expressed during hypoxia. Under normoxic conditions, HIF-1 subunits are very unstable and are rapidly targeted for degradation in the proteasome [23, 24]. Exposure to hypoxia results in a rapid increase of HIF-1 protein in most cells [25] in vivo and in vitro [14]. The HIF-1ß subunit, also called ARNT (arylhydrocarbon nuclear translocator), is constitutively expressed and unregulated by oxygen tension [26].
Although the oxygen-sensing process leading to an increase of HIF-1 protein in response to hypoxia has not been established, the mechanism by which HIF-1 protein is upregulated by hypoxia has been well characterized [13, 14]. When intracellular oxygen reaches a critically low threshold, HIF-1 subunits are rapidly protected from proteosomal degradation, allowing HIF-1 and HIF-1ß subunits to associate and form active HIF-1 [25]. Evidence suggests that HIF-1 may be stabilized under hypoxia through oxidative modifications by ROS [14]. Conversely, under normoxia, recent studies have demonstrated that proline hydroxylation in the oxygen-dependent degradation domain of HIF-1 mediates HIF-1 binding to the von Hippel-Lindau (pVHL) tumor suppressor protein. In turn, pVHL assembles a complex with E3 ubiquitin ligase that targets HIF for polyubiquitination and subsequent proteosomal degradation [23, 24].
Many investigators are studying the molecular biology of HIF-1 during embryogenesis, tumor vascularization and progression, and ischemic processes of clinical importance [19, 27]. A better understanding of the role of HIF-1 may lead to genetic therapeutic approaches for the treatment of ischemia-related diseases, including testicular ischemia. We hypothesize that HIF-1 may play an important role in the response to hypoxia in the testis following ischemic injury. To further an understanding of the molecular events involved in ischemic injury to the testis, the purpose of this study was to investigate the expression of HIF-1 mRNA and protein in the normoxic rat testis and other male reproductive organs and to determine if HIF-1 expression is affected by experimental ischemia of the testis.
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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 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 Institutes of Health Public Health Service Policy on Humane Care and Use of Laboratory Animals, 1996.
Animals were anesthetized with halothane (Sigma-Aldrich, St. Louis, MO), and surgeries were performed via a midline laparotomy using sterile procedures. Experimental ischemia was created by placing a ligature of 4-0 silk suture around the spermatic artery for 15 min, 30 min, 1 h, 3 h, or 6 h to produce a range of hypoxic time points. Unilateral surgeries were performed because preliminary studies demonstrated that even slight reductions in tissue perfusion resulting from anesthesia induces HIF-1 protein in contralateral sham-operated testes. For 1, 3, and 6 h surgeries, rats were allowed to recover from anesthesia prior to being sacrificed. Qualitative scoring of the degree of ischemia was based on evaluating the appearance of the ischemic testis compared with the sham-operated control testis as described by Turner [5]. Hypoxic kidneys were used as control tissues to detect HIF-1 protein. Kidney hypoxia was created by placing a ligature around the renal artery for 1 h prior to removing the organ. Rats were killed with carbon dioxide, and organs were dissected free of fat and then placed into ice-cold phosphate-buffered saline for nuclear protein extractions. Portions of tissue were also immediately frozen in liquid nitrogen and stored at -70°C prior to RNA isolation.
Experimental testicular torsion surgeries were carried out through a midline laparotomy essentially as described by Turner et al. [5]. These surgical conditions have been demonstrated to produce torsion-induced ischemia-reperfusion injury to the rat testis. The gubernaculum was cut in the middle to create testicular and scrotal segments for reconnection. Connective tissue holding the testis to the epididymis was separated and the testis rotated 720° counterclockwise. The incision was closed and the testis maintained in torsion for 1 h. After 1 h, the testis was detorted, the testicular stump of the gubernaculum was sutured to the scrotal stump of the gubernaculum using 4-0 silk suture and the testis and epididymis returned to the scrotum for 1 h of reperfusion prior to tissue collection as described above.
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) complementary to the rat HIF-1 gene (GenBank accession no. AF057308) were designed using JellyFish software (v1.1) and synthesized by MWG-Biotech (High Point, NC). These primers amplify a 209-base pair (bp) fragment of the rat HIF-1 gene.
HIF-1 was coamplified by relative reverse transcription polymerase chain reaction (RT-PCR) with either mouse 18S rRNA primers and competimers (Ambion, Inc., Austin, TX) or mouse ß-actin (Stratagene, La Jolla, CA) primers as internal controls. Primer and competimer concentrations were optimized (2:8 ratio) to amplify control PCR products in the same linear range as HIF-1 amplicons. The 18S and ß-actin primers amplified products of 489 and 514 bp, respectively.
One microgram of total RNA was reverse transcribed and amplified by the one-step Access RT-PCR procedure (Promega Corporation, Madison, WI) in a 50-µl reaction volume containing 50 pmol of each HIF-1 primer and either 50 pmol of each 18S primer or 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, and 40 cycles of PCR was performed 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 and stained with ethidium bromide. RT-PCR controls routinely run included a single primer pair positive control amplification, no RNA template (negative control), RNase-treated negative control, and a no reverse transcriptase negative control.
HIF-1 RT-PCR products were verified by Southern blot analysis using a human HIF-1 cDNA (GenBank accession no. U22431) probe. Human HIF-1 cDNA was provided by Dr. Christopher A. Bradfield (McArdle Laboratory for Cancer Research, Madison, WI). HIF-1 cDNA was random primed labeled with digoxigenin-dUTP using a DIG High Prime DNA Labeling system and hybridized to Southern blots of RT-PCR products according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN). Probe hybridized to HIF-1 was immunodetected with a 1:10 000 dilution of sheep anti-digoxigenin-alkaline phosphatase Fab-fragments followed by incubation with the chemiluminescence substrate CSPD and exposure to x-ray film (Kodak BioMax ML).
Nuclear Protein Extraction and Immunoblot Analysis
Nuclear and cytoplasmic protein extracts were prepared from fresh unfrozen decapsulated testes using Pierce NE-PER extraction reagents according to the manufacturer's instructions (Pierce, Rockford, IL). A cocktail of protease inhibitors and reducing agents containing aprotinin (2 mg/ml), dithiothreitol (0.5 M), leupeptin (2 mg/ml), pepstatin (2 mg/ml), and PMSF (2 M) was added to each reagent. Protein concentrations were determined by the Bradford assay (Bio-Rad, Hercules, CA) using BSA as the standard. One hundred-microgram aliquots of nuclear proteins were separated by denaturing SDS-PAGE using 7.5% polyacrylamide gels (Bio-Rad) according to the method of Laemmli [28]. HIF-1 and 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 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 1:100 dilution of C-19 HIF-1 goat polyclonal IgG (SC-8711; Santa Cruz Biotechnology, 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). 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 Incorporated, Littleton, CO). 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 bovine anti-goat IgG (Santa Cruz) conjugated to horseradish peroxidase (HRP) to detect HIF-1 or goat anti-rabbit IgG-HRP to detect HIF-1ß or 1:10 000 dilution of sheep anti-mouse IgG-HRP (Novus Biologicals, Inc.) to detect -tubulin. Blots were washed extensively in three changes of 1x Western wash for 35 min, 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 (Kodak BioMax ML).
Dephosphorylation Assays and Phosphoprotein Immunoblots
One hundred-microgram aliquots of nuclear proteins from hypoxic testes were subjected to dephosphorylation assays using 
phosphatase (-PPase) as previously described by Richard et al. [29]. Protein aliquots were lyophilized to dryness and resuspended in 1x -PPase buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM Na2 EDTA, 5 mM dithiothreitol, 0.01% Brij 35, and 2 mM MnCl2) provided by the manufacturer (New England Biolabs, Beverly, MA). Dephosphorylation reactions were carried out in the presence of 400 U of -PPase for 1 h at 30°C, then proteins were subjected to SDS-PAGE and immunoblotting. Immunoprecipitations were carried out on 100-µg aliquots of nuclear protein from hypoxic testes using a Seize X Protein G Immunoprecipitation kit and HIF-1 polyclonal antibody or HIF-1 monoclonal antibody (NB 100–105; Novus Biologicals, Inc.) according to the manufacturer's instructions (Pierce). Immunoprecipitated HIF-1 was subjected to SDS-PAGE and immunoprobed with HIF-1 polyclonal antibody or a 1:100 dilution of mouse monoclonal anti-phosphoserine antibodies (Sigma-Aldrich), a 1:1000 dilution of mouse monoclonal anti-phosphothreonine antibodies (Sigma-Aldrich), or a 1:1000 dilution of mouse monoclonal anti-phosphotyrosine antibodies (Santa Cruz Biotechnology, Inc.). Blots were incubated with goat anti-mouse IgG-HRP, then washed and developed by enhanced chemiluminescence as described above. BSA conjugated to phosphoserine, phosphothreonine, and phosphotyrosine were used as positive controls. Preincubation of primary antibodies with phosphothreonine, phosphoserine, or phosphotyrosine peptides prior to immunoblotting was also used as a control.
Quantitation of Results and Statistical Analysis
RT-PCR gel images and autoradiograms from immunoblots were digitized using a flatbed scanner, and densitometric quantitation of results was carried out with ONE-DScan software (Scanalytics, Inc., Fairfax, VA). Integrated peak areas for HIF-1 PCR products were normalized to integrated peak areas for either 18S rRNA or 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 Normoxic Tissues
HIF-1 mRNA was detected by relative RT-PCR analysis in all organs of the male reproductive tract in normoxic rats (Fig. 1). Quantitation of RT-PCR experiments revealed relatively even expression of HIF-1 mRNA in the testis, all segments of epididymis, ductus deferens, seminal vesicles, prostate, and penis (Fig. 2). No statistically significant differences in HIF-1 mRNA expression in these tissues were detected (P < 0.05). These results demonstrate that HIF-1 mRNA is widely expressed in all male reproductive organs with a level of expression equivalent to that of kidney (positive control), a tissue previously reported to be an abundant source of HIF-1 mRNA [22].
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Expression of HIF-1 mRNA and Protein in the Hypoxic Testis
RT-PCR analysis of total RNA from testes following ischemia created by clamping the spermatic artery for 15 or 30 min or for 1, 3, and 6 h showed expression of HIF-1 mRNA at all times of ischemic injury (Fig. 3). Immunoblot analysis of nuclear protein extracts from normoxic testes revealed very faint expression of HIF-1 protein in a majority of experiments, but in some experiments, HIF-1 protein was not detected in the normoxic testis (Fig. 4A). In both the normoxic and the ischemic testis, a HIF-1 protein doublet was detected with relative migrations of 78 and 90 kDa (Fig. 4A). As expected, HIF-1ß was detected as a 93-kDa polypeptide in the normal and hypoxic testis (Fig. 4B).
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In the ischemic testis, HIF-1 protein was elevated compared with the normoxic testis. Within 15 min of ischemic injury, HIF-1 protein levels showed a statistically significant (P < 0.05) increase over protein levels in the normoxic testis and rapidly reached an average 2-fold increase by 30 min (Fig. 5). Quantitation of HIF-1 mRNA expression at all time periods of ischemic treatment showed no statistically significant changes compared with normoxic testes (P < 0.05; Fig. 5). HIF-1ß protein was constitutively expressed following all times of ischemic injury (Fig. 5).
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Expression of HIF-1 Following Experimental Testicular Torsion
To determine if ischemic injury created by experimental testicular torsion stimulates HIF-1 protein expression, as was observed following ligation of the spermatic artery, rats were subjected to 720° unilateral testicular torsion for 1 h (ischemia) or testicular torsion for 1 h followed by 1 h of reperfusion (ischemia-reperfusion). As shown in Figure 6, HIF-1 mRNA expression was unchanged after 1 h of torsion-induced ischemia or 1 h of ischemia-reperfusion injury compared with normoxic controls (Fig. 6, A and C). HIF-1 protein expression was significantly increased after 1 h of torsion-induced ischemia (P < 0.05) compared with normoxic controls, and protein expression remained elevated following 1 h of ischemia-reperfusion injury (Fig. 6, B and C). HIF-1ß protein expression was not affected by ischemia or ischemia-reperfusion (Fig. 6, B and C).
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Testicular HIF-1 Is Not a Phosphoprotein
To investigate if the HIF-1 doublet detected on immunoblots of nuclear proteins from normoxic and hypoxic testes represented phosphorylated variants of HIF-1, immunoprecipitated HIF-1 from hypoxic testes was dephosphorylated with the nonspecific protein phosphatase, -PPase, and subjected to immunoblotting with HIF-1 antibodies or anti-phosphoprotein antibodies (Fig. 7). Treatment with -PPase did not change the relative molecular masses of testicular HIF-1, and the HIF-1 doublet persisted following dephosphorylation (Fig. 7A). Furthermore, immunoprecipitated testicular HIF-1 was not recognized by anti-phosphoprotein antibodies for phosphoserine, phosphothreonine, and phosphotyrosine (Fig. 7B). Taken together, these results suggest that testicular HIF-1 is not a phosphoprotein.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The RT-PCR experiments clearly demonstrate that HIF-1 mRNA is highly expressed throughout the male reproductive tract under physiologic oxygen concentration. Highly vascularized tissues such as the kidney have been reported to be a rich source of HIF-1 [31]. Our studies show that HIF-1 mRNA expression in the rat kidney equals that of the normoxic testis and all male rat reproductive organs. It was not surprising to find expression of HIF-1 throughout male reproductive organs because mRNA for HIF-1 and HIF-1ß is ubiquitously expressed in most mammalian tissues [22].
Previously, in an analysis of tissue-specific expression of HIF-1, other investigators had demonstrated that HIF-1 is expressed in the mouse testis [22, 31]; however, an extensive analysis of HIF-1 expression in other male reproductive organs was not conducted. To our knowledge, our work is the first comprehensive examination of HIF-1 expression throughout the rat male reproductive tract. Widespread expression of HIF-1 in the male reproductive tract suggests that HIF-1 may be involved in the response to ischemia in these tissues. Studies are underway to examine the role of HIF-1 in the epididymis.
Testicular HIF-1 protein was detected in the normoxic and hypoxic testis as a doublet that migrated with an apparent mass of approximately 78 and 90 kDa, and this doublet was not observed in control tissues such as the kidney, which showed HIF-1 at 90 kDa. The appearance of a doublet for HIF-1 was somewhat surprising. The molecular mass of the HIF-1 polypeptide in most mammalian tissues approximates 91–120 kDa in size [18], although it has been reported that in vitro translation of the mouse HIF-1 cDNA produces a polypeptide of approximately 86 kDa [31]. Although the amino acid sequence of human, mouse, and rat HIF-1 share approximately 95% identity, the mouse HIF-1 gene contains a first exon (mHIF-1 I.1) that can be alternatively spliced to express a mRNA encoding a HIF-1 isoform (mHIF-1 I.2) lacking the first 12 amino acids present in human HIF-1 [22, 32, 33]. Recent studies by Marti et al. [34] detected isoform-specific transcripts of HIF-1 in the mouse testis. In situ hybridization analysis suggested seminiferous tubule stage-specific expression of the exon I HIF-1 (mHIF-1 I.1) in elongating spermatids. Marti et al. also reported that expression of mHIF-1 I.1 protein appeared to be oxygen independent while 6-h exposure to whole animal hypobaric hypoxia induced expression of exon 2 (mHIF-1 I.2) protein in spermatocytes and Sertoli cells [34]. It is possible that rat testicular HIF-1 may include both isoforms of HIF-1.
Other investigators have reported hypoxia-induced phosphorylated isoforms of HIF-1, with similar variations in molecular mass as detected in our study [29, 35]. In some tissues and certain types of cultured cells, HIF-1 is known to be phosphorylated on serine residues [35] and hypoxia-induced phosphorylation of HIF-1 has been shown to be dependent on a number of kinases [29, 36], but active HIF-1 is not phosphorylated in all tissues. To examine the possibility that the testicular HIF-1 doublets represent phosphorylated isoforms of HIF-1, dephosphorylation experiments and anti-phosphoprotein immunoblots were carried out. Results from these studies suggest that testicular HIF-1 is not a phosphoprotein.
There are several other possible explanations for the testicular forms of HIF-1. HIF-1 is known to undergo extensive and varied posttranslational modifications [13]. It is possible that these doublets represent hydroxylated forms of HIF-1 [23, 24, 37], glycosylation variants, nitrosylation variants, partially ubiquinated polypeptides of HIF-1 [38], or products of slight proteolysis. It is also possible that testis-specific mRNA isoforms or animal-strain allelic variants are being expressed. Additional studies are underway to further characterize testicular HIF-1 and to determine which cells in the rat testis are producing HIF-1.
Results from experiments in which the spermatic artery was tied closed to produce ischemia and experimental torsion ischemia and ischemia-reperfusion experiments demonstrated that steady-state amounts of HIF-1 protein in the ischemic testis increased without an increase in HIF-1 mRNA while HIF-1ß protein was unregulated by hypoxia. Immunoblot detection of HIF-1 in the hypoxic testis was increased within 15 min of ischemia, and this response reached a plateau by 30 min. These findings are consistent with previous work in a number of tissues demonstrating that HIF-1 protein is rapidly stimulated by decreasing concentrations of oxygen while HIF-1ß is a constitutive protein [13, 17]. Increases in HIF-1 protein are not mediated by transcriptional or translational regulatory mechanisms and, in a majority of mammalian tissues examined, HIF-1 mRNA expression is unaffected by hypoxia. It has been well established that the hypoxic up regulation of HIF-1 protein and down regulation under normoxia is controlled through changes in protein stability [13, 17]. HIF-1 is polyubiquitinated and degraded in the proteasome in normoxic cells [14]. Recent studies have demonstrated that the pVHL 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 [23, 24].
Given the large body of work that clearly demonstrates that an increase in HIF-1 protein under hypoxic conditions is regulated by controlling ubiquitination and degradation of HIF-1, it is likely that the increase of HIF-1 protein in the ischemic testis is due to stabilization of HIF-1 from proteosomal degradation. Results from our testicular torsion ischemia-reperfusion studies provide further evidence for the oxygen-dependent nature of testicular HIF-1 protein. No statistically significant changes in expression of HIF-1 mRNA were detected following 1 h of 720° torsion-induced ischemia or 1 h of ischemia-reperfusion injury. However, HIF-1 protein was elevated by 1 h of ischemia and remained elevated following 1 h of ischemia-reperfusion. Because HIF-1 is rapidly degraded under normoxic conditions, it was expected that HIF-1 protein levels might return to normal following reperfusion. However, because HIF-1 protein remained elevated following reperfusion, this result suggests that oxygen levels in the torsion-induced ischemic testis do not return to normal or that HIF-1 is not fully destabilized under these conditions.
Several studies have provided evidence that HIF-1 protein may be stabilized during hypoxia through mechanisms that involve oxidative modifications by reactive oxygen species (ROS) [14, 39]. This is interesting, given that a role for ROS and apoptotic pathways in germ-cell apoptosis following ischemia-reperfusion of the testis has been demonstrated [5, 10]. It has been hypothesized that leukocytes may infiltrate the ischemic testis and release ROS, leading to germ-cell-specific apoptosis [5]. Increases in leukocyte diapedesis and intratesticular lipid peroxidation products likely to result from damage by ROS following 1 h of ischemia-inducing torsion injury have been reported [5]. A decrease in neutrophil infiltration of the ischemic testis in E-selectin knockout mice has been observed, suggesting that E-selectin-mediated pathways are required for leukocyte diapedesis [12].
The stimulation of HIF-1 protein observed in this study occurs within a time period of ischemic injury previously reported by other investigators. Rat testicular blood flow and interstitial oxygen tensions have been shown to dramatically decrease following 720° torsion for 1 h, with a return to physiological conditions within 7 days of torsion repair [40]. Turner et al. [5] noted significant increases in germ-cell apoptosis at 24 h after torsion repair. An investigation of apoptotic pathways following ischemia-reperfusion injury of the rat testis revealed upregulation of mRNA expression for caspases and Bax, variable upregulation of Bcl-XL and Bcl-X5, and no changes in expression of mRNA for Fas or Bcl-2 [10]. It is clear that many of the cellular and molecular mechanisms involved in germ-cell death following testicular torsion and ischemia-reperfusion injury involve apoptotic pathways. We speculate that HIF-1 is involved in torsion injury through multiple pathways. There is sufficient evidence to suggest that some of these mechanisms may involve ROS-mediated events and/or apoptotic pathways.
HIF-1 is known to specifically activate transcription of dozens of genes involved in oxygen homeostasis by binding to DNA via the hypoxia response element consensus sequence 5'-RCGTG-3' [13, 41]. To consider potential target genes for HIF-1 in the testis, an analysis of upstream promoter sequences for testis genes expressed in the human, rat, or mouse testis containing the HIF-1 consensus DNA binding site were identified using Genomatix MatInspector software (v3.0.03; http://genomatix.gsf.de/matinspector). Our survey revealed several genes with one or more consensus DNA binding sites for HIF-1, suggesting the regulation of important target genes by testicular HIF-1.
Of particular interest, we identified genes known to be expressed in the testis that are involved in apoptosis, including cytochrome C [10], and p53 [42], anti-apoptotic pathways (Bcl-2) [43], ROS metabolism (SOD-1) [44], and angiogenesis (VEGF) [43], that may be potential candidate gene targets for HIF-1 in the testis. Although Bcl-2 does not appear to be affected by testicular ischemia [10], it is intriguing that several genes involved in apoptosis may be targets for HIF-1 in the ischemic testis. In the case of p53, HIF-1 has been shown to increase p53 expression in hypoxic cells leading to apoptosis [20], and HIF-1 may interact directly with p53 to stabilize p53 [45, 46]. Possible convergence of HIF-1, ROS pathways, and apoptotic mechanisms during testicular ischemia warrants further investigation.
Other potential candidate genes identified included hexokinase [47] and the transferrin receptor [48], a HIF-regulated gene that may play a role in iron-dependent signaling of oxygen tension [49]. It is possible that alterations in germ-cell metabolism together with, or independent of, apoptotic events contribute to germ-cell loss in the ischemic testis. We are interested in addressing whether HIF-1 contributes to germ-cell loss via activation of apoptotic pathways or if HIF-1 plays an unsuccessful role in trying to rescue germ cells from hypoxia by attempting to activate genes involved in oxygen homeostasis.
Results presented in this study strongly support a role for HIF-1 in the cellular and molecular response to hypoxia during testicular ischemia. Additional studies are necessary to further characterize testicular HIF-1, elucidate the role(s) of HIF-1 in the ischemic testis, and examine possible associations of HIF-1 with cellular mechanisms involved in apoptosis and germ-cell loss associated with testicular ischemic injury.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Monmouth University Biology Department and a TriBeta Research Foundation Scholarship to D.J.F. This study was partially presented at the VIIth International Congress of Andrology in Montréal, Canada, June 2001. 
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
Accepted: April 22, 2002.
Received: February 19, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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in rat and human prostate cancer. Cancer Res 1998 58:5280-5284 (HIF-1) and enhance the transcriptional activity of HIF-1. J Biol Chem 1999 274:32632-32637 and HLF/EPAS, two basic loop helix/PAS domain transcription factors involved in adaptive responses to hypoxic stresses. Biochem Biophys Res Commun 1997 240:552-556[CrossRef][Medline] protein expression is controlled by oxygen-regulated ubiquitination that is disrupted by deletions and missense mutations. Proc Natl Acad Sci U S A 2000 97:4748-4753. Nature 1998 392:405-408[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  T. T. Turner and J. J. Lysiak Oxidative Stress: A Common Factor in Testicular Dysfunction J Androl, September 1, 2008; 29(5): 488 - 498. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Kazi and R. D. Koos Estrogen-Induced Activation of Hypoxia-Inducible Factor-1{alpha}, Vascular Endothelial Growth Factor Expression, and Edema in the Uterus Are Mediated by the Phosphatidylinositol 3-Kinase/Akt Pathway Endocrinology, May 1, 2007; 148(5): 2363 - 2374. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Zhang, O. W. Moe, J. A. Garcia, and C. C. W. Hsia Regulated expression of hypoxia-inducible factors during postnatal and postpneumonectomy lung growth Am J Physiol Lung Cell Mol Physiol, May 1, 2006; 290(5): L880 - L889. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. T. Turner, J. J. Lysiak, J. D. Shannon, Q. A. T. Nguyen, and C. R. Bazemore-Walker Testicular Torsion Alters the Presence of Specific Proteins in the Mouse Testis as Well as the Phosphorylation Status of Specific Proteins J Androl, March 1, 2006; 27(2): 285 - 293. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Lidgren, Y. Hedberg, K. Grankvist, T. Rasmuson, J. Vasko, and B. Ljungberg The Expression of Hypoxia-Inducible Factor 1{alpha} Is a Favorable Independent Prognostic Factor in Renal Cell Carcinoma Clin. Cancer Res., February 1, 2005; 11(3): 1129 - 1135. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Depping, S. Hagele, K. F. Wagner, R. J. Wiesner, G. Camenisch, R. H. Wenger, and D. M. Katschinski A Dominant-Negative Isoform of Hypoxia-Inducible Factor-1{alpha} Specifically Expressed in Human Testis Biol Reprod, July 1, 2004; 71(1): 331 - 339. [Abstract] [Full Text] [PDF]
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M.A. Palladino, J.D. Powell, N. Korah, and L. Hermo Expression and Localization of Hypoxia-Inducible Factor-1 Subunits in the Adult Rat Epididymis Biol Reprod, April 1, 2004; 70(4): 1121 - 1130. [Abstract] [Full Text] [PDF]
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