Animals and surgical procedures
Adult male, retired-breeder Sprague–Dawley rats (550–650 g) purchased from Charles River Laboratories (Stone Ridge, NY) were housed under controlled conditions of temperature and humidity on a twelve-hour light/dark cycle. Animal care and surgical procedures were carried out following guidelines established by the National Institutes of Health (NIH) Public Health Service Policy and the Monmouth University Institutional Animal Care and Use Committee (IACUC).
Animals were anesthetized using injections of sodium pentobarbital (Sigma-Aldrich, St. Louis, MO) at a concentration of 75 mg of pentobarbital per kg of body weight. Torsion surgeries were performed via a midline laparotomy using sterile procedures and with animals maintained on warming pads to prevent hypothermia as previously described [8, 13]. Torsion was induced unilaterally to create an experimental state of ischemia. Following an abdominal incision, the gubernaculum was cut and the testis and epididymis were exposed through the laparatomy incision. The connective tissue holding the testis to the epididymis was separated and the testis rotated 720° clockwise. Then, the testis and epididymis were returned to the scrotum, the laparatomy incision closed and the testis maintained in torsion. In reperfusion experiments, each torsed testis was detorsed and the testicular and scrotal stumps of the divided gubernaculum were sutured together to hold the testis in place for the duration of the reperfuson treatment phase and the testis and epididymis were returned to the scrotum.
Contralateral, sham-treated testes served as controls. The surgeries were carried out on alternating sides so that sham or ischemia induction was performed in right and left testes for all time points. Each animal had a sham testis and an ischemic testis. Animals were maintained under anesthesia for the duration of the surgical treatment.
The treatment groups involved the following conditions (n = 3–6 animals/group): 1 hour or 6 hours of ischemia (I), or 1 hour of ischemia followed by 4 hours of reperfusion (1h/4h I/R). At the conclusion of the treatment, euthanasia was performed via carbon dioxide asphyxiation in accordance with the Report of the American Veterinary Medical Association (AVMA) Panel on Euthanasia (2000). The testes were excised, then frozen in liquid nitrogen and stored at −70°C prior to RNA or protein isolation. Apoptosis of germ cells in the testes following I and I/R was evaluated via terminal deoxynucleotidyltransferase mediated dUTP-biotin nick end labelling (TUNEL) assays to validate cell damage. These data were previously reported in Supplemental Data of Palladino et al.  and demonstrated an increase in germ cell-specific apoptosis following I and I/R, but no statistically significant changes in apoptosis of Leydig cells after I and I/R.
Isolation of cytoplasmic and nuclear proteins
Fresh or frozen tissue samples were placed on ice and homogenized in 3 volumes of lysis buffer composed of 10 mM Tris–HCl (pH 7.5), 1.5 mM MgCl2, and 10 mM KCl with freshly added 1 mM dithiothreitol (DTT), 1 mM Na3VO4, and 10 mM protease inhibitor mix (P8340; Sigma-Aldrich, St. Louis, MO). The homogenate was centrifuged at 3,500 rpm at 4°C for 5 minutes and the resulting supernatant was saved as the cytoplasmic fraction and stored at −80°C. To isolate nuclear proteins, the pellet obtained from the initial centrifugation step was resuspended in 0.42 M KCl, 20 mM Tris–HCl (pH 7.5), 1.5 mM MgCl2, and 20% glycerol, with freshly added 1 mM dithiothreitol (DTT), 1 mM Na3VO4, and protease inhibitor cocktail (P8340; Sigma-Aldrich), mixed at 150 rpm at 4°C for 30 minutes to liberate proteins from nuclei and then centrifuged at 13,500 rpm for 30 minutes at 4°C. The resulting supernatant containing nuclear proteins was removed and stored at −80°C. Protein concentrations were determined via the Bradford assay (Bio-Rad, Hercules, CA).
Enzyme-linked immunosorbent assay (ELISA) to detect HIF-1 DNA binding
Testicular HIF-1 DNA binding was determined using the Active Motif TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif, Carlsbad, CA) following the manufacturer’s instructions. In this assay, wells are pre-coated with oligonucleotides containing the consensus hypoxia response element (HRE: 5’-TACGTGCT-3’, consensus sequence for the HRE underlined) from the human EPO gene and HIF-1 DNA binding of protein extracts is determined by incubating 10 μg aliquots of nuclear proteins in the wells for 1 hour at room temperature. Protein extracts from CoCl2-treated hypoxic COS-7 cells (Active Motif) were used as positive controls for HIF-1 binding. After several washing steps, 100 μl of a goat anti-HIF-1α primary antibody (AF1935; R&D Systems, Inc., Minneapolis, MN) was added, followed by incubation for 1 hour at room temperature and detection with an anti-IgG-horseradish peroxidase (HRP)-conjugated secondary antibody. Colorimetric changes were determined via spectrophotometric measurement of the absorbance at 450 nm, with a reference reading obtained at 655 nm and a background calculation deducted from each well. The absorbance readings were quantitated as a fold increase of the negative control. Samples were run in triplicate using protein extracts isolated from testes treated for different times under I or I/R (n = 3–6 animals for each treatment group) and the statistical significance of results was determined via Analysis of Variance (ANOVA).
Electrophoretic mobility shift assays (EMSA)
Electrophoretic mobility shift assays were performed using the Thermo Scientific LightShift® Chemiluminescent EMSA kit according to the manufacturer’s instructions (Thermo Scientific, Rockford, IL). The reactions were carried out in 20 μl volumes with 5 μg of a nuclear or cytoplasmic protein extract from the testis incubated with 10× binding buffer, 1 μg/μl poly (dI·dC), 50% glycerol, 1% NP-40, 1M KCl, 100 mM MgCl2, and 200 mM EDTA and biotin-labelled EMSA oligonucleotides for 20 minutes at room temperature. Epstein-Barr Nuclear Antigen (EBNA) and biotin-labelled double-stranded target DNA was included as a positive control for the EMSA. A double-stranded HIF-1 oligonucleotide containing the consensus HRE (5’-TCTGTACGTGACCACACTCACCTC-3’, consensus sequence for the HRE underlined; sc-2625, Santa Cruz Biotechnology, Santa Cruz, CA) and mutant oligonucleotides (sc-2626; Santa Cruz Biotechnology) with an AAA substitution in place of the CGT in the consensus site were used for EMSA.
Terminal deoxynucleotidyl transferase (TdT) was employed to catalyze the biotin labelling of the oligonucleotides used for EMSA according to the manufacturer’s instructions (Pierce Biotin 3’ End DNA Labelling Kit). Labelling reactions with a 50 μl volume containing the appropriate oligonucleotides were incubated for 30 minutes at 37°C in ultrapure water, 5X TdT Reaction Buffer, Biotin-11 UTP, and diluted TdT. To stop each reaction, 0.2 M EDTA was added and 24:1 chloroform: isoamyl alcohol extraction performed to remove protein from labelled oligonucleotide. Labelled oligonucleotides were stored at −20°C.
The EMSA reactions were separated using 6% polyacrylamide TBE PAGEr® Gold gels (Lonza, Rockland, ME) electrophoresed in 0.5× (0.89 M) Tris-borate-EDTA buffer (Lonza AccuGENE® 10× TBE Buffer). Binding reactions were transferred onto nylon membrane (Thermo Scientific Biodyne® B) which were then cross-linked at 120 mJ/cm2 for 60 seconds using a UV-light cross-linker instrument. The blots were blocked with blocking buffer, incubated with a 1:300 dilution of a stabilized streptavidin-HRP conjugate for 15 minutes at room temperature and then reacted with luminal/enhancer solution (Pierce Chemiluminescent Nucleic Acid Detection Module). The blots were exposed to x-ray film (BioMax ML; Kodak, Rochester, NY) for 2–5 minutes, or digital images were captured using a ChemiDoc™ XRS+ Molecular Imager with Image Lab™ Software (Bio-Rad).
For competition experiments, an excess of the unlabeled HIF-1α consensus oligonucleotide was used to demonstrate competition for DNA binding between biotin-labelled and unlabeled oligonucleotides. In supershift experiments, the specificity of HIF-1 DNA binding was demonstrated on 4-20% polyacrylamide TBE PAGEr® Gold gels which were electrophoresed in 0.5× Tris-borate-EDTA (Lonza). Goat anti-human polyclonal antibodies C-19 and Y-15 (sc-8711 and sc-12542 respectively; Santa Cruz Biotechnology) were used to perform supershift experiments. All EMSA experiments were replicated a minimum of 5 times with independent samples (n=3-6 animals for each treatment group). Representative results are presented.
Immunoblot analysis was performed following denaturing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) of nuclear and cytoplasmic samples in 7.5% or 10% PAGEr® Gold Tris-Glycine polyacrylamide gels (Lonza, Rockland, ME). Tissue extracts obtained from COS-7 cells (Active Motif, Carlsbad, CA), rat PC-12 pheochromocytoma (Active Motif), and the BJAB human B cell lymphoma line (Santa Cruz Biotechnology, Santa Cruz, CA) were included as positive controls for detecting HIF-1. Seventy-five microgram aliquots of protein samples were heated for 5 minutes at 95°C prior to loading and then electrophoresed at 150 V for approximately 90 minutes. Proteins were electroblotted onto Trans-Blot nitrocellulose membranes (Bio-Rad), and Ponceau S staining (0.0005% in 1% acetic acid) was used to confirm protein transfer. The blots were blocked in 1× Western Wash (50 mM Tris, pH 7.6, 30 mM NaCl, 0.001% Tween 20) containing 5% nonfat dry milk under gentle agitation at room temperature for 30 minutes. For the detection of MCL-1, the blots were incubated overnight with gentle agitation at 4°C in a 1:1,000 dilution of an Mcl-1 primary antibody (rabbit polyclonal IgG;C. sc819, Santa Cruz Biotechnology) in 1× Western Wash and 5% nonfat dry milk.
After 3 washes with 1× Western Wash, the blots were incubated with an HRP-conjugated secondary antibody (goat anti-rabbit IgG; sc2054) at a 1:20,000 dilution in 1× Western Wash with 5% nonfat dry milk for 1 hour at room temperature under gentle agitation. Following 3 washes with 1×7 Western Wash, the blots were developed via enhanced chemiluminescence in SuperSignal West Pico or Femto substrate (Pierce, Rockford, IL) and exposed to X-ray film (Biomax ML).
The blots were stripped of bound antibody by incubation in Restore Buffer (Pierce) at 37°C for 15 minutes and then washed in 1× Western Wash for 5 minutes, prior to blocking and reprobing. The blots were probed for activating transcription factor-2 (ATF-2) using a 1:1,000 dilution of an anti-ATF-2 antibody (rabbit polyclonal IgG; sc-6233) to detect ATF-2 as a hypoxia-independent loading control for protein quantification. Negative control experiments to determine antibody specificity were carried out by incubating the blots with pre-immune sera, followed by incubation with secondary antibodies. Mcl-1 protein levels were normalized to ATF-2 as a loading control. Immunoblot data were analyzed by one-way ANOVA and the results considered significantly different at P< 0.05.
Immunoblot analysis was conducted to determine the presence of MCL-1 in cultured Leydig cells. Cultured cells were provided by the Hardy lab of the Population Council at the Center for Biomedical Research (New York, NY). Briefly, Leydig cells were purified from 325 to 350 g Sprague–Dawley rats using a previously described collagenase dispersal and sedimentation approach . Typically, 6 animals were required to yield 10 to 20 million adult Leydig cells. A hemocytometer was used to estimate cell yields and the percentage of purity was assessed via histochemical staining to detect 3β-hydroxysteroid dehydrogenase with etiocholan-3β-ol-17-one as the enzyme substrate. The adult Leydig cell preparations used for these experiments were over 95% pure. Equal number of cells were allocated to T-25 flasks and cultured in buffered Dulbecco’s modified Eagle’s medium: F12 culture medium supplemented with ovine luteinizing hormone (0.1 ng/mL) and lipoproteins (Lipimate; Hyclone Logan, Utah). Fresh isolates of cells were used for isolating MCL protein, or cells were cultured at 34° C for 20 hours in an incubator containing either a 5% O2/5% CO2 atmosphere or a 21% O2/5% CO2 atmosphere. Five separate replicates were performed for each of the culture conditions in these experiments. Leydig cells were collected from the flasks using trypsin, centrifuged and frozen in liquid nitrogen as cell pellets for subsequent MCL protein isolation.The applied oxygen concentrations were chosen to mimic hypoxic and normoxic conditions, respectively.
Cytoplasmic extracts were isolated and separated on 10% Tris-glycine polyacrylamide gels via SDS-PAGE and immunoprobing procedures were carried out as described above. The immunoblots were run with the total protein aliquots isolated from equivalent numbers of cells (1 million cells) for each treatment group.
To determine which cell types in the testis express Mcl-1, tissue sections were prepared for immunocytochemistry. Tissues were placed in 10% (w/v) neutral-buffered formalin (Richard-Allan Scientific, Kalamazoo, MI) overnight prior to paraffin embedding. The tissue samples were then dehydrated in three successive changes of 70%, 95% and 100% ethanol then transferred through three changes of xylene and incubated overnight at 60°C in a 1:1 xylene/molten paraffin mixture. Finally, the samples were incubated at 60°C in fresh molten paraffin for 1 hour through a total of 3 changes and then embedded in paraffin. Paraffin sections were cut at a thickness of 5 μm and mounted on glass slides (Superfrost® Plus; VWR).
The sections were deparaffinized in xylene and hydrated through a series of graded ethanol solutions and then rinsed in 1× phosphate-buffered saline (PBS) solution for 5 minutes to complete the hydration steps. Then, the slides were covered in antigen unmasking solution (Vector Laboratories, Inc., Burlingame, CA), microwaved on high for 10 minutes and cooled for 1 hour at room temperature. The slides were next rinsed in 1× PBS for 5 minutes and quenched in 0.5% (v/v) H2O2/methanol for 30 minutes, then washed in 1× PBS for 5 minutes, placed into a humidified chamber, and incubated with blocking serum for 20 minutes using serum from the species of origin of the secondary antibody. A 1:100 dilution of an Mcl-1 primary antibody (rabbit polyclonal IgG; sc819) in 1× PBS was added to the testis samples as well as slides containing sections of spleen as a positive control, and the slides incubated in a humidified chamber overnight at 4°C. Negative control sections were incubated in the absence of primary antibody.
The slides were washed in 1× PBS for 5 minutes with a total of 3 changes. The slides were then incubated in biotinylated secondary antibody (goat anti-rabbit IgG; sc2054) at a 1:200 dilution in 1× PBS combined with Santa Cruz blocking serum for 45 minutes in a humidified chamber at room temperature. Next, the slides were washed in 1× PBS for 5 minutes through a total of 3 changes and incubated in the Santa Cruz AB (avidin-biotin) enzyme reagent for 30 minutes at room temperature. The slides were subsequently washed in 1× PBS through a total of 3 changes, and sections were incubated with diaminobenzidine/peroxidase substrate for 10 minutes at room temperature followed by washing with dH2O prior to counterstaining with hematoxylin. Finally, the sections were dehydrated in a graded series of ethanol solutions, placed in xylene, and cover slips were mounted using Permount (Fisher Scientific, Pittsburgh, PA). The slides were examined under a Nikon Eclipse 50i light microscope (Nikon Instruments, Inc., Melville, NY) and photographed using a DS-L1 digital camera.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were carried out with the Active Motif ChIP-IT™ Express kit as per protocols outlined in the manufacturer’s instruction manual. In place of cultured cells, a 0.50 g sample of freshly excised testis from sham and surgically treated animals was used to isolate chromatin complexes. The tissue samples were incubated in 20 ml of fixation solution (37% formaldehyde; Dulbecco’s modified Eagle’s medium) under gentle agitation for 10 minutes at room temperature, then washed with ice-cold 1× PBS. Fixation was stopped by adding 10 ml of a glycine-1× PBS stop-fix solution followed by swirling and rocking at room temperature for 5 minutes. The samples were washed with 1× ice-cold PBS and cells were pelleted by centrifugation at 2,500 rpm for 10 minutes at 4°C. Finally, 1 μl of 100 mM phenylmethylsulfonylfluoride (PMSF) and 1 μl protease inhibitor cocktail (PIC) were added to the cell pellets, which were then stored at −80°C.
For ChIP assays, the pellets were subsequently thawed in 1 ml of ice-cold lysis buffer (supplemented with 5 μl PIC + 5 μl PMSF) and incubated on ice for 30 minutes. Cell nuclei were released by homogenizing the cell pellets using an ice-cold glass dounce homogenizer followed by centrifugation at 5,000 rpm for 10 minutes at 4°C to pellet chromatin. The pellets were resuspended in 1.0 ml of digestion buffer pre-warmed at 37°C and supplemented with 5 μl PIC + 5 μl PMSF.
Chromatin DNA was enzymatically sheared using DNase (200U/ml) and optimal enzymatic shearing conditions determined by placing 50 μl aliquots of chromatin in digestion buffer that had been pre-warmed at 37°C for 5 minutes and adding a working stock of an enzymatic shearing cocktail to the chromatin, followed by incubation for 5, 10 or 15 minutes. The reactions were stopped by adding 1 μl of ice-cold 0.5 M EDTA followed by chilling on ice for 10 minutes. Sheared chromatin samples were then centrifuged at 10,000 rpm for 10 minutes at 4°C and stored at −80°C. Proteinase K-treated chromatin samples were electrophoresed through 1% agarose gels prepared in 1× sodium boric (SB) acid buffer (Faster Better Media, LLC, Baltimore, MD)  containing SYBR® Safe (Invitrogen) and visualized to determine the size range of the sheared DNA and optimum shearing conditions.
A magnetic bead system was employed to capture HIF-1/chromatin complexes. Briefly reaction tubes were loaded with Protein G magnetic beads, ChIP Buffer, sheared chromatin, PIC, and 100 μg of a HIF-1α primary antibody (AF1935; R&D Systems, Inc., Minneapolis, MN) diluted in 3 μl of 1× PBS in 100 μl reaction volumes. Chromatin in positive control samples was incubated with a primary antibody for RNA polymerase II (ChIP-IT™ Express Enzymatic, Active Motif), and negative control samples were incubated with a nonspecific IgG antibody or polyclonal antibody to β-actin. The reactions were incubated on a rolling shaker for 4 hours at 4°C, and the tubes were then placed on a magnetic stand to pellet the beads on the side of the tube. The supernatant was discarded and the beads were washed three times with ChIP buffers and reverse cross-linking buffer to elute chromatin.
Polymerase chain reaction (PCR) amplification was employed to identify ChIP-enriched sequences. The PCR master mix consisted of the following components: 196 μl of diethylpyrocarbonate-treated H2O, 50 μl 10× PCR buffer, 50 μl 10× loading dye, 20 μl of a dNTP mixture, 4 μl of Taq polymerase, and 80 μl of dH2O (for control PCR sample only) or 40 μl forward/40 μl reverse primers (either for β-actin or for Mcl-1). The rat β-actin primer came from the ChIP-IT™ Control Kit (Active Motif). Mcl-1 forward (5’-ACTTGAGGCCATGAGTTCGAGACCA-5’) and reverse (5’-CTCCACTTCCCACGTTCAGACGATT-3’) primers were designed based on the rat Mcl-1 promoter sequence (AF147742.1) spanning the HRE portion of the promoter and were purchased from MWG Biotech Inc. (High Point, NC).
Aliquots of chromatin samples were incubated in PCR mix containing either β-Actin primers or Mcl-1 primers, and the samples were amplified using the following thermocycling conditions: 36 cycles of 94°C for 20 seconds, 59°C for 30 seconds, and 72°C for 30 seconds. Analysis of the obtained PCR products was carried out via electrophoresis of 8 μl aliquots of each amplified sample on 2% agarose gels prepared in 1× SB buffer containing SYBR® Safe. The PCR products were cloned into pGEM-T Easy plasmid vectors (Promega Corp., Madison, WI) and sequences of enriched DNA were confirmed by cycle sequencing using a LI-COR 4300L DNA sequencer (LI-COR Biosciences, Lincoln, NE) and BLAST (Basic Local Alignment Search Tool analysis (Altschul, et al., 1990).