All animals were maintained and handled according to the policies approved by CHA University Institutional Animal Care and Use Committee (IACUC, approval number 170002). Eight-week-old adult ICR mice were provided by Orient Bio, Inc (Gapyeong, Gyeonggi, Korea).
To examine the actions of ovarian steroid hormones on expression of Pla2g10, adult female mice were OVX, rested for 14 days, and then appropriately treated with steroid hormones for each experiment performed in this study. Mice were sacrificed and their uterine horns were collected for real-time RT-PCR and/or immunofluorescence after ovarian steroid hormone treatment.
To investigate time-dependent actions of P4 (Sigma-Aldrich, USA) and E2 (17b-estradiol, Sigma-Aldrich) on the expression of Pla2g10 in mouse uterus, adult OVX mice were subcutaneously injected with P4 (2 mg/mouse) or P4 + E2 (200 ng/mouse) and sacrificed at various time points (0, 3, 6, and 24 h) after injection. To examine the dose-dependent induction of Pla2g10 by P4, mice were given a single injection of vehicle (sesame oil, 0.1 ml/mouse) or P4 at various concentrations (0.25 to 2 mg). To analyze whether P4 works through a nuclear PR for Pla2g10 expression in mouse uterus, adult OVX mice were pretreated with the PR antagonist RU-486 (1 mg/mouse, Sigma-Aldrich), 30 min before P4 (2 mg/mouse) injection and then sacrificed 24 h later.
Preparation of uterine samples during early pregnancy
Uterine samples during early pregnancy were prepared as previously described . Briefly, 8- to 10-week-old female mice were housed with proven fertile males for pregnancy. The next morning when the vaginal plug was found was considered as Day 1. Pregnant mice were sacrificed on various days of pregnancy, and their uteri were collected for real-time RT-PCR and/or immunofluorescence. IS in the morning (0900 h) of Day 5 and 6 were visualized by intravenous injection (0.1 ml/mouse) of Chicago sky blue 6B solution (1% in saline, Sigma-Aldrich). The IS were demarcated by discrete blue bands along the uterus. IS on Day 6 were collected and immediately frozen in liquid nitrogen for frozen section to perform histological analyses including immunofluorescence staining and alkaline phosphatase (ALP) activity assay.
To induce an experimentally-induced delayed implantation model in mice, pregnant ICR female mice were OVX at the morning of Day 4 and given P4 (2 mg/mouse) from Day 5 to 7 as described previously . To activate dormant blastocysts and initiate implantation, P4-primed delayed implanting pregnant mice were injected with E2 (25 ng/mouse) on Day 7. Mice were sacrificed 24 h after the last hormone injection, and IS were visualized using Chicago sky blue 6B solution.
RNA extraction, RT-PCR, and real-time RT-PCR
The experiment was performed as previously described . Briefly, uteri (3-5 mice per each group) were collected and immediately frozen in liquid nitrogen. Then, total RNA was extracted individually using Trizol Reagent (Ambion, USA) according to manufacturer’s protocols. cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Promega, USA) with random primers and oligo dT. Synthesized cDNA was utilized for PCR with specific primers at optimized cycles. Real-time RT-PCR was performed by monitoring real-time increases in the fluorescence of SYBR Green dye. Real-time RT-PCR was performed using the Realtime PCR detection system (Bio-Rad, USA) and iQTMSYBR® Green supermix (Bio-Rad). For comparison of transcript levels between samples, a standard curve of cycle thresholds for several serial dilutions of a cDNA sample was established and then used to calculate the relative abundance of each gene. Values were then normalized to the relative amounts of rPL7 cDNA. All PCR reactions were performed in duplicate.
To determine the presence and cell-type specific localization of PLA2G10 after P4 treatment, and during the estrous cycle and early pregnancy, uteri were fixed in 4% paraformaldehyde (PFA) and embedded in paraplast (Leica Biosystems, Germany). Uterine sections (5 µm) were deparaffinized, dehydrated, and subjected to antigen retrieval in 0.01 M sodium citrate buffer, pH 6.0, for 20 min. For immunofluorescence staining of ARG2 (Arginase 2), a marker for decidualization, frozen sections (13 µm) of IS on Day 6 were fixed in 4% PFA, washed in PBS, and permeabilized with 0.1% triton-X 100 in PBS. Non-specific staining was blocked using Protein Block Serum-Free (Dako, Denmark) for 1 h. Then, sections were incubated overnight with primary rabbit-anti-PLA2G10 antibody (1:100, Santa Cruz Biotechnology, USA) for PLA2G10 or primary rabbit-anti-ARG2 antibody (1:200, abcam, USA) for ARG2 at 4 °C, washed in phosphate-buffered saline (PBS), and incubated with Alexa Fluor 488 goat-anti-rabbit secondary antibody (1:1000, Invitrogen Corp., USA) for 1 h at room temperature. Sections were washed in PBS, counterstained with propidium iodide (PI, Sigma-Aldrich) for 20 min, and mounted for observation using a LSM880 confocal microscope (Carl Zeiss, Germany).
Hematoxylin & Eosin (H&E) staining and ALP activity assay
H&E staining and ALP activity assay were performed to evaluate gross histology of implanted embryos and decidualization in IS of the uterus with siPla2g10 on Day 6, respectively. Frozen sections (13 µm) were fixed in 4% PFA, washed in PBS, and either stained with hematoxylin (Cancer Diagnostics, USA) and eosin (Richard Allan Scientific, USA) or incubated with a 100mM Tris HCl buffer (pH 9.5) containing ALP substrate working solution (Vector Laboratories, SK-5400, USA). Slides were counterstained with fast red and mounted to observe ALP activity under light microscopy.
Construction of expression and reporter vectors
A proximal region (-840 to +65) of Pla2g10 promoter (p) was amplified from mouse genomic DNA by PCR with Forward 1 (5'-GCT AGC GGT GGT TCC AAG GTT TCA CTC AG-3') and Reverse 1 (5'-CTC GAG GTC ACA GAG GTG GCC CAC AC -3') primers. The amplified Pla2g10(p) was cloned into pGL4.10 vector (Promega) and named pGL4.10/Pla2g10(p)-840/+65. The vector was independently mutated at four PREs, namely -801/-794, -356/-349, -310/-303, and -290/-283 in Pla2g10(p)-840/+65 using the EZ change™ Site-directed Mutagenesis Kit (Enzynomics, Inc., Korea). The four mutated PREs were named pGL4.10/Pla2g10(p)-801mt, pGL4.10/Pla2g10(p)-356mt, pGL4.10/Pla2g10(p)-310mt, and pGL4.10/Pla2g10(p)-290mt, respectively. PRA and PRB cDNAs were provided by Dr. J.W. Jeong (Michigan State University, MI, USA). The cDNAs were cloned into a pcDNA3.1 NheI-XhoI site and named pcDNA3.1/PRA and pcDNA3.1/PRB, respectively.
Transfection and luciferase assay
Ishikawa cells, human endometrial adenocarcinoma cells, were plated in 12-well plates with DMEM/F12 and 10% charcoal-stripped (CS)-FBS 24 h before transfection. pcDNA3.1, pcDNA3.1/PRA, or pcDNA3.1/PRB expression vectors were co-transfected with pGL4.10/Pla2g10(p)-840/+65, pGL4.10/Pla2g10(p)-801mt, pGL4.10/Pla2g10(p)-356mt, pGL4.10/Pla2g10(p)-310mt, or pGL4.10/Pla2g10(p)-290mt vectors, and a pRL-null vector that was used as an internal control for normalization by GenePORTER®3000 Transfection Reagent (Genlantis, USA). The medium was replaced with DMEM/F12 and 2% CS-FBS with 1 µM P4 (Sigma-Aldrich) 4 h after transfection. Cells were harvested and analyzed for firefly and renilla luciferase activities using the Dual-Glo™ Luciferase Assay System (Promega) 24 h after transfection. Luminescence was measured with Synergy Mx™ (Bio Tek, Inc., USA).
In vivo RNA interference of Pla2g10 in mouse uterus
Knock-down of Pla2g10 in mouse uterus was performed as previously described by Ruan et al. with some modifications . Briefly, 100 pmol siPla2g10 (BIONEER Corp., Korea; 5'-GAA CAA AUG CCA AGA ACU U-3’) or siNC (BIONEER Corp.) were combined with 5 µl of lipofectamine 2000 in 10 µl of Opti-MEM. The solutions were injected into each uterine horn at 18:00 - 20:00 h on Day 3 for in vivo RNA interference of Pla2g10 in mouse uterus.
All values represent the mean ± standard deviation. The unpaired Student’s t-test was used for statistical evaluation. A p-value of less than 0.05 was considered statistically significant.