Collection of CL tissue during PGF-induced luteolysis
The animal procedures were approved by the local institutional animal care and use committee of Polish Academy of Sciences in Olsztyn, Poland (Agreement No. 5/2007, 6/2007 and 88/2007). Healthy normally cycling Polish Holstein Black and White cows were used for collection of CL. Estrus was synchronized in the cows by two injections of an analogue PGF (Dinoprost, Dinolytic; Pharmacia & Upjohn, Belgium) with an 11-days interval. Ovulation was determined by a veterinarian via transrectal ultrasonography examination. Then, CLs were collected by Colpotomy technique using a Hauptner’s effeninator (Hauptner and Herberholz, Solingen, Germany) on day 10 post-ovulation, i.e., just before administration of a luteolytic dose of analogue PGF (Dinoprost, Dinolytic; Pharmacia & Upjohn, Belgium) (0 h), and at 0.5, 2, 12 and 24 h post-treatment (n=4 per time-point). CL tissues were dissected from the ovary and then stored at −80°C until the SOD1 mRNA, protein and total SOD activity analysis.
Bovine LEC isolation and cell culture
LECs were isolated from five CLs at the mid-luteal phase (days 8–12 of the estrous cycle)  using magnetic beads as previously described  and recently validated in our laboratory [6, 18]. Briefly, magnetic tosylactivated beads (Dynabeads M-450, 140.04; Dynal ASA, Oslo, Norway) were coated with 0.15 mg/ml lectin from Bandeiraea simplicifolia (BS-1; L2380; Sigma-Aldrich, St. Louis, MO, USA), which specifically binds the glycoproteins expressed by bovine LECs . Luteal cell (LC) suspension was mixed with beads at a concentration of 4 × 108 beads/ml, and incubated for 20 min at 4°C on a rocking platform. The cells were pooled and cultured in endothelial cell (EC) growth medium (MV 2; C22121; Promo Cell, Heidelberg, Germany) at 37°C in a humidified atmosphere of 5% CO2 in air. Only colonies with a homogeneous cell population were removed with a pipette and cultured in collagen-coated 25 cm2 culture flasks (690175; Greiner Bio-One, Frickenhausen, Germany). The cultures and passages were repeated until a homogeneous population of pure LECs was obtained. The LECs used in the present study were previously confirmed to be positively stained with rabbit anti-human von Willebrand factor (vWF, F3520; Sigma-Aldrich), isolated LECs expresses CD31 but not 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA as reported previously [6, 17, 18]. Experiments were performed on confluent cultures and the cells (LECs) were from passage 2–5.
The LECs were seeded at a concentration of 1 × 105 viable cells/ml into 24-well plates (662160; Greiner Bio-One) for determination of SOD1 mRNA or PGF production, and 1 × 106 viable cells/ml into 75-cm2 culture flasks (658175; Greiner Bio-One) for determination of SOD1 protein expression and total SOD activity in culture cells. LECs were cultured in DMEM/F-12 (D/F; D8900; Sigma-Aldrich) supplemented with 10% calf serum (v/v, 16170–078; Life Technologies Inc., Grand Island, NY, USA), 20 μM gentamicin (G1397; Sigma-Aldrich) and 2 μM amphotericin B (A9528; Sigma-Aldrich). The cells reached confluence on 5th day of culture. After the cells reached confluence, the medium was replaced with fresh D/F supplemented with 5 μg/ml holo-transferrin (T3400; Sigma-Aldrich), 500 μM ascorbic acid (013–12061; Wako Pure Chemical Industries, Ltd., Osaka, Japan), 5 nM sodium selenite (S5261; Sigma-Aldrich), and 0.1% (w/v) BSA (10 735 078 001; Roche Diagnostics, Mannheim, Germany).
Cell viability test
LECs cultured in 96-well plates were exposed to H2O2 (0.1 – 1000 μM) for the final 24 h of culture. The cell viability was determined by Dojindo Cell Counting Kit including WST-1 (Dojindo, Kumamoto, Japan; No. 345–06463). Briefly, WST-1, a kind of MTT [3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide], is a yellow tetrazolium salt that is reduced to formazan by live cells containing active mitochondria. The culture medium was replaced with 100 μl D/F without phenol red medium-BSA, and a 10-μl aliquot (0.3% WST-1, 0.2 mM 1-methoxy PMS in PBS, pH 7.4) was added to each well. The cells were then incubated for 4 h at 38°C. The absorbance (A) was read at 450 nm using a microplate reader (Bio-Rad, Hercules, CA; Model 450). Percentage of cytotoxicity was determined by subtracting the mean A of H2O2-treated wells (Atest) from the mean A of untreated wells (Acontrol) and then dividing by the mean A of untreated wells (Acontrol). The mean A of wells in the absence of the cells was subtracted from the mean A of all experimental wells. The percent cytotoxicity was calculated as 100 × (Acontrol – Atest)/(Acontrol).
Dose-dependent effect of PGF and H2O2 on SOD1mRNA expression at 2 h in vitro
To determine whether LECs were responsive to increased concentrations of PGF and H2O2 in the present culture system, LECs cultured in 24-well plates were incubated for 2 h with or without 0.1-10 μM PGF or 1–100 μM H2O2 (n=4 experiments). The cells were then collected and stored at −80° C for the analysis of SOD1 mRNA expression.
SOD1 protein and total SOD activity at 2 h and at 24 h in vitro
To examine whether SOD is differently regulated by PGF and H2O2 at the time of functional and structural luteolysis, LECs cultured in 75-cm2 culture flasks were incubated for 2 h (mimicking functional luteolysis) and 24 h (mimicking structural luteolysis) with or without PGF (1 μM) or H2O2 (10 μM). The samples were then used for analysis of SOD1 protein expression and total SOD activity (n=4 experiments). The concentrations of PGF and H2O2 were chosen based on the result of SOD1 mRNA expression in vitro, and the effect of H2O2 on cell viability
PGF production in vitro
To determine the dose-dependent effects of H2O2 on PGF production, LECs cultured in 24-well plates were exposed to H2O2 (1–100 μM) for 2 h or 24 h. After incubation, the conditioned media were collected in 1.5 ml tubes containing 5 μl of a stabilizer solution (0.3 M EDTA, 1% (w/v) acid acetyl salicylic, pH 7.3) and frozen at −30°C until the PGF assay (n=4 experiments).
Total RNA was prepared from the CL tissue or cultured bovine LECs using TRI reagent according to the manufacturer’s direction (TRI Reagent RNA isolation protocol, © 2008 Ambion, Inc). One microgram of total RNA of each sample was reverse transcribed using a SuperScript First-Strand Synthesis System for RT-PCR (11904–018; Invitrogen), and the reaction mixture was used in each PCR together with the appropriate oligonucleotide primer pairs. The primer for SOD and beta-actin (ACTB) were designed and characterized as described previously . Primer for SOD1 was: forward: AAGGCCGTCTGCGTGCTGAA; reverse: CAGGTCTCCAACATGCCTCT (accession No: M81129; product: 240 bp). Primer for ACTB was: Forward: CGGCATTCACGAAACTACC; Reverse: ATCAAGTCCTCGGCCACAC (accession No: AY141970; product: 536).
The RT-PCRs were conducted with the house-keeping gene ACTB as an internal standard. ACTB primer was added at the appropriate cycle number by the “primmer-dropping method” as described by Wong et al.  with modification . The PCRs were carried out using TaKaRa Taq (R001A; Takara Bio Inc., Shiga, Japan) and a thermal cycler (iCycler; Bio-rad Laboratories, Hercules, CA, USA). The PCR conditions were as follow: activation of DNA polymerase for 20 sec at 95°C, annealing for 1 min at 60°C, and extension for 1 min at 70°C, follow by final extension for 5 min at 72°C. The number of cycles was 27 for SOD and 23 for ACTB. Two-fifths aliquot of each reaction mixture was electrophoresed on a 1.5% agarose gel containing ethidium bromide with a known standard (100-bp ladder, New England Biolabs Inc., Beverely, MA, USA; #N3231S) and photographed under ultraviolet illumination. The integrated density was determined by ImageJ software (Windows version of NIH Image, http://rsb.info.nih.gov/nih-image/, National Institutes of Health). Relative density was quantified by normalization of the integrated density of each corresponding β-actin.
SOD1 protein expression
SOD1 protein expression in luteal tissue and in cultured bovine LECs was assessed by Western immunoblotting analysis. Tissues or cells were lysed in 150 μl lysis buffer (20 mM Tris–HCl, 150 mM NaCl, 1% Triton X-100 [Bio-Rad Laboratories], 10% glycerol [G7757; Sigma-Aldrich], Complete [11 697 498 001; Roche Diagnostics, Basel, Switzerland], pH 7.4). Protein concentrations in the lysates were determined by the method of Osnes et al. , using BSA as a standard. The proteins were then solubilized in SDS gel-loading buffer (10% glycerol, 1% β-mercaptoethanol [137–06862; Wako Pure Chemical Industries, Ltd.], pH 6.8) and heated at 95°C for 10 min. Samples (50 μg protein) were electrophoresed on a 15% SDS-PAGE for 1.5 h at 30 mA. The separated proteins were electrophoretically transblotted to a 0.2-μM nitrocellulose membrane (LC2000; Invitrogen) at 300 mA V for 3 h in transfer buffer (25 mM Tris–HCl, 192 mM glycine, 20% methanol, pH 8.3). The membrane was washed in TBS-T (0.1% Tween 20 in TBS [25 mM Tris–HCl, 137 mM NaCl, pH 7.5]), incubated in blocking buffer (5% nonfat dry milk in TBS-T) for 1 h at room temperature, incubated at 4°C with a primary antibody specific to each protein (goat SOD1 polyclonal antibody [23 kDa; 1:500 in TBS-T, overnight; sc-8637; Santa Cruz Biotechnology, Santa Cruz, CA, USA] and mouse ACTB monoclonal antibody [internal standard, 42 kDa; 1:4000 in TBS-T, overnight; A2228; Sigma-Aldrich]). The membrane was washed three times for 5 min in TBS-T at room temperature, incubated with secondary antibody (SOD1 [1:10000 in TBS-T]: anti-goat Ig, HRP-linked whole antibody produced in donkey, sc-2020; Santa Cruz; ACTB [1:40000 in TBS-T]: anti-mouse Ig, HRP-linked whole antibody produced in sheep, NA931; Amersham Biosciences, Buckinghamshire, UK) for 1 h, and washed three times in TBS for 5 min at room temperature. The signal was detected by a ECL Western immunoblotting detection system (RPN2109; Amersham Biosciences). The intensity of the immunological reaction was estimated by measuring the optical density in the defined area by computerized densitometry using NIH Image (National Institutes of Health; Bethesda, MD, USA).
Total SOD activity assay
Total SOD activity was determined in CL tissues collected at 0, 0.5, 2, 12, and 24 h after PGF injection and in the LECs at the end of a 2-h or 24-h incubation period using SOD Assay Kit-WST (S311-08; Dojindo Laboratories, Kumamoto, Japan). Total SOD activity was calculated using a concurrently run SOD standard curve, and expressed as inhibition rate or percentage of control (raw data on total SOD activity was normalized based on protein concentrations, units per mg of cellular protein).
Determination of PGF concentration
The concentration of PGF in the culture medium was determined by enzyme immunoassay (EIA) as described previously . The PGF standard curve ranged from 15.625 to 4000 pg/ml, and the median effective dose (ED50) of the assay was 250 pg/ml. The intra- and inter-assay coefficients of variation were 7.4 and 11.6%, respectively. The cross-reactivities of the antibody were 100% for PGF, 3.0% for PGD2, 1.1% for PGI, 0.15% for PGE2, and < 0.03% for PGA2. The DNA content, estimated using the spectrophotometric method by Labarca & Paigen , was used to standardize the PGF concentrations.
Data of SOD1 mRNA, protein level and total SOD activity were obtained in four separate experiments. PGF concentration and cell viability were performed in triplicate samples for each experimental group of LECs or CL tissues (in vivo). The statistical significances of differences in the amounts of SOD1 mRNA, protein levels, total SOD activity, cell viability and differences in PGF concentrations were analyzed using two-way analysis of variance (ANOVA) with repeated-measures or one-way ANOVA followed by Fisher’s protected least-significant difference (PLSD) procedure as multiple comparison tests. All values were expressed as the mean ± SEM of four separate experiments. A level of P<0.05 was considered to be statistically significant.