Animals
Adult male, retired-breeder Sprague-Dawley rats (475–750 g, 9–12 months, Charles River Laboratories (Stone Ridge, NY) were housed individually on a 12:12-h light/dark cycle and controlled temperature with free access to food and water.
Administration of Lipopolysaccharides
P. aeruginosa LPS (Type 10, L7018; Sigma-Aldrich, St. Louis, Missouri) and E. coli 055:B5 LPS (L2880; Sigma-Aldrich) were selected because both are from known pathogens of the urogenital tract and cause tissue-specific inflammatory responses. A dosage of 5.0 mg/kg body weight was chosen to maximize activation of inflammatory responses. Others have clearly demonstrated LPS doses that generate inflammatory responses in vivo (1–5 mg/kg body weight) and in vitro (0.1–1.0 mg/ml), models [10, 16, 18, 19, 32]. LPS was reconstituted in sterile 1× phosphate buffered saline (PBS; 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.8) and administered via intraperitoneal injection. Sham controls were injected with sterile PBS. After 1, 3, 6, and 12 h of treatment (n = 5–7/time point), animals were euthanized via CO2 gas. Testes and control tissues were excised then frozen in liquid nitrogen and stored at − 80 °C.
Serum testosterone assays
Blood was collected via cardiac puncture at the time of sacrifice, clotted at room temperature for 30 min, centrifuged at 13,000 rpm for 10 min at 4 °C then sera was removed and stored at − 80 °C prior to testosterone measurements. Testosterone radioimmunoassays were carried out using a Packard 1900TR Liquid Scintillation Analyzer (Canberra, Meriden, CT). Internal controls included a no testosterone control and a positive control sample of 100 ng/ml testosterone.
Protein extraction
Frozen tissue samples were homogenized on ice in three volumes of lysis buffer (10 mM Tris-HCL [pH 7.5], 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM Na3VO4 containing protease inhibitor cocktail; P8340; Sigma-Aldrich). The homogenate was maintained on ice for 10 min then centrifuged for 5 min at 3500 rpm at 4 °C. Supernatant was removed for cytoplasmic proteins. Nuclei were resuspended in three volumes of 0.42 M KCl, 20 mM Tris-HCL (pH 7.5), 1.5 mM MgCl2, 20% glycerol, mixed for 30 min at 150 rpm at 4 °C, and centrifuged at 13,500 rpm for 30 min at 4 °C to isolate nuclear proteins. Protein extracts were stored at − 80 °C and protein concentrations determined by the Bradford assay (Bio-Rad, Hercules, CA).
Immunoblot analysis
Proteins were separated by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) through 7.5% PAGEr® Gold Precast Gels (Lonza, Rockland, MD) in 1× Tris-glycine SDS buffer (0.25 mM Tris, 192 mM glycine, and 0.1% (w/v) SDS [pH 8.3]). COS-7 simian virus 40-transformed kidney cell nuclear extract (Active Motif, Carlsbad, CA) was included as a positive control for detecting HIF-1α. RAW 264.7 cell extract (Santa Cruz Biotechnology, Santa Cruz, CA) was included as positive control for NF-κB and IκBα. Proteins were electroblotted onto Trans-Blot nitrocellulose (Bio-Rad), blocked with 1× Western wash (50 mM Tris, 30 mM NaCl, 0.001% Tween 20 [pH 7.6]) containing 5% nonfat dry milk (NFDM) for 30 min at room temperature, then incubated in primary antibody overnight at 4 °C in 1× Western wash, 5% NFDM.
Primary antibody dilutions were as follows: 0.5 μg/mL HIF-1α mouse monoclonal antibody (AF1935; R&D Systems, Minneapolis, MN), 0.5 μg/mL NF-κB rabbit polyclonal antibody (ADI-KAP-TF112, Enzo Life Sciences, Farmingdale, NY), 0.5 μg/mL IκB rabbit polyclonal antibody (9242; Cell Signaling, Danvers, MA). Actin was detected as a loading control for protein quantification using a 1:2000 dilution of rabbit polyclonal antibody (A2066, Sigma-Aldrich). Blots were incubated in 1× Western wash, 5% NFDM containing appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:20,000 dilution), washed in 1× Western wash, developed by enhanced chemiluminescence using either SuperSignal® West Pico substrate or SuperSignal® West Femto substrate (Pierce, Rockford, IL), and analyzed with a ChemiDoc™ XRS+ Molecular Imager with Image Lab™ Software (Bio-Rad).
Gene expression profile analysis by real-time quantitative PCR (qPCR)
Total RNA was isolated from frozen tissue using TRIzol reagent (Invitrogen, Grand Island, NY). Gene expression profiles for hypoxia pathway genes and inflammatory pathway genes were analyzed using Rat Hypoxia Signaling Pathway and Rat Innate and Adaptive Immune Response Pathway RT2 Profiler™ PCR Arrays (Qiagen, Frederick, MD). One microgram of genomic-DNA-free total RNA was reverse transcribed using the RT2 First Strand cDNA Kit (330,401, Qiagen). Real-time qPCR assays were carried out with SYBR Green dye and amplified for 40 cycles in a Stratagene Mx3500P® thermocycler. Each array contained 84 genes involved in the hypoxia or innate and adaptive immune response pathways. Listings of all genes queried and accession numbers are available from the Qiagen website.
Positive controls and housekeeping genes were included to normalize for differences in sample loading, genomic DNA contamination, and amplification efficiency. PCR controls included no RT and no template negative controls. Relative quantification of all target genes was calculated by first normalizing comparative cycle threshold (Ct) values of target genes to ß-actin. Normalized values were used to calculate target gene expression in treatment groups compared to shams. Ct values of controls were ~ 8–10 cycles beyond RT test samples indicating that no contaminating DNA was present. Genes that displayed an average (n = 4–7) minimum of three-fold change (up- or down-regulation) were considered statistically significant (p < 0.05) by analysis of variance (ANOVA).
In silico analysis
Computer-based (in silico) methods were utilized for the analysis of candidate genes involved in inflammatory responses of the testis. Methods included literature searches using PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) and bioinformatics databases such as UniProt (https://www.uniprot.org) for gene expression and protein distribution data. These electronic resources were used to determine if there was existing data available about these genes, such as expression and regulation data, cell-type-specific expression in the testis, and protein expression data from other researchers. Information about each gene of interest was gathered to propose pathway maps. The Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources 6.7 available through the National Institute of Allergy and Infectious Diseases (NIAID) was utilized for functional annotation and pathway map analysis (https://david.ncifcrf.gov).
Electrophoretic mobility shift assays
Electrophoretic Mobility Shift Assays (EMSA) were performed using the Thermo Scientific LightShift® Chemiluminescent EMSA kit according to manufacturer’s instructions (Thermo Scientific, Rockford, IL). Terminal deoxynucleotidyl transferase (TdT) was used to catalyze biotin labeling of oligonucleotides (Biotin 3′ End DNA Labeling Kit, Pierce). Binding reactions were carried out in 20 μl volumes with protein extracts incubated with 10× binding buffer, 1 μg/μl poly (dI•dC), 50% glycerol, 1% NP-40, 1 M KCl, 100 mM MgCl2, and 200 mM EDTA and biotin-labeled oligonucleotide for 20 min at room temperature. Epstein-Barr Nuclear Antigen (EBNA) and biotin-labeled double-stranded target DNA were included as positive controls. Unlabeled EBNA oligonucleotides were used in negative control and competition experiments. NF-κB double-stranded oligonucleotide with a consensus binding site for NF-κB /c-Rel homodimeric and heterodimeric complexes (5’-AGTTGAGGGGACTTTCCCAGGC -3′; sc-2505, Santa Cruz Biotechnology) which is similar to the sequence present in the upstream region of the HIF-1 gene and mutant oligonucleotides (sc-2511) with a C-G substitution in the consensus site were used for EMSA.
EMSA reactions were separated through 6% polyacrylamide TBE PAGEr® Gold gels (Lonza) electrophoresed in 0.5× tris-borate-EDTA buffer (AccuGENE® 10× TBE Buffer, Lonza), transferred onto nylon (Biodyne® B, Thermo Scientific) and membranes cross-linked at 120 mJ/cm2 for 60 s using a UV-light cross-linker. Blots were blocked, incubated with a 1:300 dilution of stabilized streptavidin-HRP conjugate for 15 min at room temperature, then reacted with luminal/enhancer solution. Digital images were captured with a ChemiDoc™ XRS+ quantitative analysis performed using Image Lab™ Software.