Patients
MGCs samples aspirated during IVF procedures in IVF unit in the Sheba Medical Hospital were used. The study was approved by the local Institutional Review Board (IRB) committee of Chaim Sheba Medical Center, Tel Hashomer (ethical approval number SMC-11-8707 and SMC-12-9342). Written informed consent was obtained from each patient who provided samples. All experiments involving mice were conducted in compliance with the principles of the National Research Council (NRC) and were approved by the institutional animal care and use committee (IACUC) #919/14/ANIM.
A total of 33 women were included with an average age of 32±4 (mean±SD), BMI 21.5±2.3, oocyte retrieved 10±3. All patients were treated with antagonist protocols to get consistent results and avoid the effect of different protocols (see below)
In Vitro Fertilization (IVF) Protocol
Patients underwent ovarian stimulation using a GnRH antagonist protocol (Cetrorelix; Merck Serono, Darmstadt). Ovarian stimulation was performed using a daily subcutaneous dose of human recombinant FSH (either Gonal-F; Merck Serono, an affiliate of Merck KGaA, Darmstadt, Germany or Puregon; Schering Plough, North Wales PA) which was commenced on the third day of the menstrual cycle. After 5 days of stimulation, the women received human menopausal gonadotrophin (hMG; Menopur, Ferring, Switzerland). The initial dose used was dependent upon age, body mass index and prior IVF treatment history. When three or more follicles exceeded 18 mm in diameter, 250 µg of hCG (Ovitrelle; Merck Serono) was administered to trigger ovulation. Oocyte retrieval was performed 36 h following hCG triggering by transvaginal ultrasound-guided needle aspiration.
Cumulus granulosa cell collection
After COC retrieval, CCs of each oocyte were removed with the use of hyaluronidase (SAGE, Trumbull, CT, USA) and a glass denudation pipette (Swemed, Billdal, Sweden). The CGCs were washed in Phosphate-Buffered Saline (PBS) and centrifuged at 5000 × g for 5 minutes at room temperature. The resulting pellets were stored at −80°C until RNA isolation. CCs of individual oocytes were classified as per the corresponding oocyte maturation stage: CCs from GV oocytes (CCGV) and CCs from MII oocytes (CCM2). CCs obtained from individual oocytes were collected from individual subjects were pooled to generate a single replicate (n=3-4 different subjects). Each experiment was performed at least three times.
Mural Granulosa Cell Collection and Grouping
MGCs were collected from the aspirated follicular fluid (Follicles size >= 17 mm) and re-suspended in a phosphate-buffered solution (PBS; Sigma-Aldrich-Aldrich, St Louis, MO, USA). After allowing the cells to settle by gravity for a few minutes, the top portion of the medium was repeatedly re-suspended and aspirated until such time that the medium proved clear. The cells were then centrifuged at 1000 rpm for 5 minutes at room temperature. The resulting pellets were stored at −80°C until RNA isolation. MGCs from 3 different subjects were pooled to generate a single replicate.
Mural Granulosa Cell Culture
MGCs were collected as described above and placed on a percoll gradient and centrifuge at 3000 RPM for 15 min. The MGCs were collected and washed with PBS, counted and plated in 24-well plates at a density of 100,000 cells/well, and incubated at 37°C in a humidified atmosphere with 5% CO2 in the air. The cells were cultured for 4 days with a daily medium replacement before hCG triggering as previously described (28). For signaling study, MGCs were pre-treated with H89 (10 μM) or LY 294002 (10 μM) for 30 minutes and then stimulated with either hCG (1 U/ml) or forskolin (FSK, 10 μM) or Phorbol-12-Myristate-13-AcetateXYZ (PMA, 10 μM) for an additional 24 hours (all chemicals from LC Laboratories, Woburn, MA, USA).
RNA Extraction and qPCR
RNA extraction and qPCR. Total RNA was extracted from MGCs or CCs using a Mini/Micro RNA Isolation I kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. RNA purity and concentration were assessed using a NanoDrop spectrophotometer (NanoDrop 2000C, Thermo Scientific Waltham, MA, USA). Total RNA (25ng) from each sample was used for cDNA synthesis by a high capacity reverse transcription kit (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer’s instructions in a 10μl total volume reaction. mRNA levels were analyzed by real-time PCR using the StepOnePlus real-time PCR system (Applied Biosystems). The real-time PCR mix contained 1μl of cDNA, fast SYBR Green Master Mix (Applied Biosystems), and specific primers for DUOX2 or another gene of interest and β-actin (housekeeping gene) in a total volume of 10μl. Cycling parameters were: 1 cycle at 95°C for 20 seconds and 40 cycles each at 95°C for 3 seconds and 60°C for 30 seconds. A melting curve analysis was performed at the end of each run to ensure a single amplicon. All samples were run in duplicates. Analysis of the qPCR results was carried out with StepOne software. Relative gene expression was calculated using the delta-delta Ct method. Details of the primers used are shown in Table S1.
Western blot
Cells harvested using 0.5 mL PBS and pelleted. Cell pellets were lysed in TNE buffer (50 mM Tris–HCl, pH 8.0, 250 mM NaCl, 2 mM EDTA, 1% NP-40, Sigma Aldrich St Louis Mo) and protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO), vortexed and incubated for 10 min on ice before removal of nuclei and debris by centrifugation. Aliquots of the clarified supernatants were used to determine protein concentration. Protein concentration was determined using the Bradford method (Protein Assay Dye Reagent, Bio-Rad, Hercules, CA). Equal amounts (50 μg) of protein were loaded and separated on SDS-Polyacrylamide gel (10% acrylamide). Proteins were then transferred onto nitrocellulose membranes. Membranes were blocked in 5% BSA in TBST (100 mL TBS 10X, 900 mL H2O, 1 mL Tween 20, Sigma Aldrich St Louis Mo) for one hour and afterward incubated with primary antibody (Ab Thermo Fisher, 1:500) overnight at 4˚C. The membranes were then treated with horseradish peroxidase-conjugated secondary antibody and developed using an enhanced chemiluminescence kit (Sigma Aldrich St Louis Mo).
Immunofluorescence
Immunofluorescence staining and confocal analysis. MGCs were seeded on coverslips. The cells were fixed with cold 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, blocked in 1% BSA and 10% normal donkey serum in PBS for 10 minutes, incubated with anti-DUOX2 antibody (Thermo Fisher) diluted 1:100 for 1 hour at room temperature, washed 3 times, stained with FITC-conjugated donkey anti-mouse antibody for 1 hour at room temperature, and washed again 6 times. The slides were mounted with coverslips using GelMount (Biomeda), and the cells were analyzed with a Leica SP5 (Leica) confocal laser scanning microscope.
Superovulation protocol
25-day old C57BL female mice were injected with 10U of Pregnant mare's serum gonadotropin (PMSG, Sigma, St. Louis, MO, USA) to stimulate follicle growth, and 48 h later with an ovulatory dose of 10U hCG that mimicking an endogenous LH surge, stimulating follicle development and ultimately ovulation 12–16 h later. The animals were sacrificed 48 h after PMSG treatment as well as 3, 6, 9, 12, 16 and 24 hours after hCG administration.
All mice were sacrificed by CO2 asphyxiation, and the ovaries were removed and either frozen or paraformaldehyde-fixed until used, or punctured to collect entrapped oocytes. Blood samples were collected at the time of euthanasia for progesterone measurement, and the number of oocytes within the ampullas of all oviducts was recorded.
DPI (Almog diagnostic 5 or 7 mg/kg) was injected at the same time as hCG to PMSG-primed mice. Mice treated with hCG and vehicle (DMSO) served as controls. Mice were sacrificed 16 hours after the administration
Mouse ovarian morphology
Fixed ovaries (4% formalin) were embedded in paraffin blocks, mounted on slides, and stained with hematoxylin/eosin. Mouse ovarian morphology was assessed by examining 4-μm serial histological sections cut by a microtome.
Measurement of progesterone concentrations
Blood samples for hormone assays in female mice were obtained at the time of euthanasia by cardiac puncture. Sera were separated from whole blood and frozen until the time of analysis. Progesterone concentrations were measured in duplicate by the American Medical Laboratories in Herzliya, Israel.
H2O2 measurement
MGCs aspirated during IVF procedures were cultured for 4 days with daily medium exchange (Medium 199 Sigma Aldrich St Louis Mo). MGCs were stimulated with hCG 1 U/ml. 15 hours after hCG administration, the medium was exchanged to DMEM medium (DMEM/F-12, no Phenol Red) 1% FBS (Invitrogen Grand Island, NY) and 1% penicillin/streptomycin (Sigma Aldrich St Louis Mo). MGCs were stimulated either with vehicle (control), 1 U/ml hCG, with or without DPI inhibitor 10 μM. CaCl2 1 mM was added to all cells.
Detection of H2O2 using the AmplexR Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen Catalog no. A22188). The reaction was started by adding a working solution of 100 μM Amplex® Red reagent and 0.2 U/mL HRP to each microplate well containing the standards, controls, and samples. The plate was incubated for 1 hour at room temperature protected from light. Fluorescence was then measured with a fluorescence microplate reader. Fluorescence emission detection at 570 nm. Background fluorescence, determined for a no-HRP control reaction, has been subtracted from each value.
Statistics
Each experiment was carried out at least three times. Data, expressed as mean ± SEM, were evaluated with Student's t-test (two-tailed) or with ANOVA for more than 2 variables using the post hoc Tukey test assuming equal variances or the Games-Howell test for unequal variances. When appropriate, Kruskal-Wallis non-parametric comparison test was used. SPSS 20 software (IBM) was used for all analyses. P values < 0.05 were considered statistically significant.