Study design
The objective of this study was to investigate the physiological role of the prostaglandin transporter ABCC4 in the ovulatory process. We used both in vitro and in vivo systems to meet this objective. This non-randomized laboratory study relied on human tissues and cells as well as on a mouse model. The study was approved by the local Institutional Review Board (IRB) committee of the Chaim Sheba Medical Center at Tel Hashomer (ethical approval #SMC-11-8707 and #SMC-12-9342). All experiments involving mice were conducted in compliance with the principles articulated by of the National Research Council (NRC) and approved by the Institutional Animal Care and Use Committee (IACUC) (approval #919/14/ANIM. The study was carried out in compliance with the ARRIVE guidelines. Mice were eeuthanatized using carbon dioxide in accordance to standard protocol.
Written informed consent was obtained from each patient who provided samples. A total of 27 women were included in the study. All patients were pretreated treated with a Gonadotropin-Releasing Hormone (GnRH) antagonist with an eye toward assuring experimental consistency (see below). The average age of the patients was 32±4 (mean±SD); the average BMI was 21.5±2.3; and the average number of aspirated oocytes was 10±3.
All methods were performed in accordance to the relevant guidelines and regulations.
IVF protocol
Normo-ovulatory young women (< 37 years of age) undergoing IVF because of male factor infertility or pre-implantation genetic diagnosis were selected for this study. Subjects afflicted with BRCA mutations, Fragile X disorder, Endometriosis, or Polycystic Ovary Syndrome (PCOS) were excluded. Ovarian stimulation was carried out as previously described 54,55. Briefly, a "short antagonist" protocol was used wherein controlled ovarian hyperstimulation with Human Menopausal Gonadotropins (HMG; Menopur®) or recombinant Follicle-Stimulating Hormone )rFSH, either Gonal-F®; Merck Serono or Puregon Pen®; Schering Plough) was initiated 3 days after the onset of menses. The initial gonadotropin dose used was dependent upon age, body mass index, and previous IVF treatment history. Ovarian suppression with a GnRH antagonist (0.25 mg/day, Cetrorelix, Cetrotide®; Serono International, SR) was initiated when the leading follicle was more than 12 mm in diameter. When three or more follicles exceeded18 mm in diameter, 250 µg of human Chorionic Gonadotropin (hCG; Ovitrelle®; Merck Serono) was administered to trigger ovulation. Transvaginal follicular aspiration was performed 36 hours later under ultrasound guidance.
Cumulus granulosa cell collection
After COC retrieval, CGCs 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. CGCs of individual oocytes were classified as per the corresponding oocyte maturation stage: CGCs from GV oocytes (CGGV) and CGCs from MII oocytes (CCMII). CGCs 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
Follicular fluid was aspirated from follicles ≥ 17 mm in each subject. The follicular fluid was centrifuged and the pelleted MGCs were re-suspended in 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 aspirated and the cells were repeatedly re-suspended until the medium proved clear. The cells were then centrifuged at 1000 rpm for 5 minutes at room temperature and the resulting pellets were stored at −80°C until RNA isolation. Total MGCs from 3 different subjects were pooled to generate a single replicate. Each experiment was performed at least three times.
Mural granulosa cell culture
Each MGCs sample was collected from the aspirated follicular fluid of follicles size ≥ 17 mm from one subject (unless specified otherwise) and re-suspended in 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 aspirated and the cells were repeatedly re-suspended until the medium proved clear and then placed on a Percoll® gradient and centrifuged 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 air. The cells were cultured for 4 days with daily medium replacement prior to hCG triggering for the indicated times.
RNA extraction and qPCR
Total RNA was extracted from MGCs or CGCs 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 ABCC4 or other 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 at 60°C for 30 seconds. A melting curve analysis was performed at the end of each run to ensure measurements were based on the amplification of the target gene. All samples were run in duplicate. 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.
RNA sequencing (RNAseq)
Global transcriptome assessments were performed on compact (CCGV) samples obtained from two women during in vitro maturation (IVM) and expanded (CCMII) cumulus cells samples obtained from three women during IVF as described in Yerushalmi et al. 2014 16. Briefly, a cDNA library was prepared according to Illumina recommendations (preparing samples for mRNA sequencing; Illumina). Cluster generation and single-end sequencing was carried out using the standard Illumina procedures for the HiSeq 2000 sequencer (Illumina). All sequenced reads were mapped and aligned to the human genome. The number of reads that overlap each of the annotated genes was counted and the differentially expressed transcripts were identified 16.
IVM protocol.
IVM cycles were carried out as previously describe 56. Briefly, sonographic assessment of the antral follicle count and of endometrial thickness was carried out on day 3 of a spontaneous menstrual cycle. The serum concentrations of estradiol and progesterone were also determined. Treatment with 150 IU/day rFSH for 3 days followed suit. After a second sonographic assessment on Day 6, 10,000 IU hCG (Pregnyl; Organon) was administered when the endometrial thickness was ≥5 mm and the leading follicle was ≥12 mm. Oocyte retrieval was carried out 36 hours later.
Animals
C57BL/6 mice were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN, USA). The mice were maintained under controlled lighting (12 hour light/12 hour dark) conditions with continuous access to food and water.
Superovulation protocol
25-day-old female mice were initially treated with 10 U of Pregnant Mare Serum Gonadotropin (PMSG, Chronogest, Intervet, Israel) to stimulate follicular growth. An ovulatory dose of hCG (10 U) (Ovitrelle®, Merck Serono, Darmstadt, Germany) was administered 48 hours later. To elucidate the role of ABCC4 in vivo, probenecid, at different concentrations (200-450 mg/kg), was administrated intra-peritoneally in the same time as hCG. To evaluate the combined effect of ABCC4 and PTGS inhibitors, probenecid (25 mg/kg) and meloxicam (10 mg/kg) or indomethacin (5 mg/kg) were administrated together intra-peritoneally at the same time as hCG.
Rescue experiments: To examine the specificity of probenecid, and the combined approach (probenecid and meloxicam) effect on ovulation, PGE2 (2mg/kg) was administrated intra-peritoneally in the same time as hCG.
Animals were sacrificed 48 hours after the initiation of PMSG treatment as well as 9 or 16, hours after hCG administration. All mice were sacrificed by CO2 asphyxiation, and the ovaries were removed and either flash frozen in liquid nitrogen, paraformaldehyde-fixed, or punctured in order 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 each oviduct was recorded.
Western blot
Cells were 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, USA) containing a protease inhibitor cocktail (Sigma–Aldrich, St. Louis, MO, USA), 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 using the Bradford method (Protein Assay Dye Reagent, Bio-Rad, Hercules, CA, USA). Equal amounts (50 μg) of protein were loaded and separated by SDS-polyacrylamide gel electrophoresis (10% acrylamide). Proteins were then transferred onto nitrocellulose membranes. Membranes were blocked in 5% bovine serum albumin (BSA) in TBST (100 mL TBS 10X, 900 mL H2O, 1 mL Tween 20, Sigma Aldrich St Louis, MO, USA) for one hour and afterwards incubated with a primary antibody against ABCC4 (M4I-10, Abcam, 1:500) or β-actin (housekeeping gene) overnight at 4°C. The membranes were then treated with a goat anti-rat IgG HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) and developed using an enhanced chemiluminescence kit (Thermo Scientific, Waltham, MA, USA) 57.
PGE2 measurements in cell culture medium and in cells
MGCs were plated in 24-well plates at a density of 200,000 cells/well and cultured as described above for 6 days. The MGCs were then treated for 24 hours with hCG (1U) in the absence or presence of MK-571 (50 µM) or probenecid (500 µM). The concentration of PGE2 in conditioned media was assessed using an enzyme immunoassay kit (Cayman Chemical). For intracellular PGE2 measurements, cells were harvested in PBS, resuspended in 50 μl of sonication buffer (0.1 M phosphate pH 7.4 containing 1 mM EDTA and 10 mM indomethacin), sonicated (7 sec x 3 on ice; CV18 Sonics) and centrifuged at 8000 rpm, for 10 min at 4ºC. Supernatants were diluted 1:5 with EIA buffer and subjected to EIA for PGE2 (Cayman Chemical).
Effect of ABCC4 siRNA on PGE2 levels
MGCs were plated in 6-well plates at a density of 250,000 cells/well and cultured as described above for 2-3 days. The transfection mixture included: (A) 9 µl Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) in 150 µl OptiMEM (Gibco by Life Technologies, Paisley, UK), and (B) 8 µl ABCC4 siRNA or Scramble siRNA (Santa Cruz Biotechnology, Dallas, TX, USA) in 150 µl OptiMEM medium (10 µM final concentration). The transfection mixture was incubated at room temperature for 5 minutes before added to the cells (300 µl) with additional 300 µl of OptiMEM medium. The cells without transfection reagent were covered with 600 µl OptiMEM. After the cells were incubated at 37°C for 4 h, an additional 1.4ml medium (DMED/F12 with 10% FCS media) was added to each well. The medium was changed 24 h later. All the groups, except for control, were cultured for 48 h post transfection and then stimulated with hCG for 24 h. The cells were then harvested and the medium was collected from one duplicate of the group for protein quantification using the Bradford assay. Extracellular PGE2 levels were assessed using a PGE2 Enzyme Immunoassay (EIA) kit. PGE2 levels were analyzed relative to protein levels. The cells in the other duplicate were subjected to RNA lysis buffer, diluted 1:200 for intracellular PGE2 levels and assessed by PGE2 EIA kit and to qPCR to determine ABCC4 mRNA expression levels.
Mouse ovary RNA isolation
Mouse ovaries were removed immediately after the mice were sacrificed, dissected from the surrounding fallopian tubes and fat tissue, and flash frozen in liquid nitrogen. Frozen ovaries were crushed by mortar and pestle and the RNA was purified using the Micro RNA Isolation I kit (Zymo Research, CA, USA) according to the manufacturer’s instructions.
Mouse ovarian morphology
Fixed ovaries (4% formalin) were embedded in paraffin, sectioned, mounted on slides, and stained with hematoxylin/eosin. Mouse ovarian morphology was assessed by examining 4-µm serial histological sections.
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.
Statistics
Each experiment was carried out at least three times. Data were expressed as mean ± standard error of the mean (SEM) and evaluated with Student's t-test with a two-tailed distribution, with two samples equaling variance, or with ANOVA for more than two variances 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. For all statistical analysis, SPSS 22 software (IBM, Armonk, NY, USA) was used. P values < 0.05 were considered statistically significant.