Cell culture and gene transfection
Human placental samples for research purposes were approved by the Institutional Review Board of CHA Gangnam Medical Center, Seoul, Republic of Korea (IACUC-190048). Briefly, PD-MSCs were isolated from the chorionic plate of the placenta as described in previous reports [16]. PD-MSCs and PD-MSCsPRL-1 were cultured in alpha-minimum essential medium (α-MEM; HyClone, Utah, USA) containing 10% fetal bovine serum (FBS; Gibco-BRL, Oklahoma, USA), 1% penicillin/streptomycin (P/S; Gibco-BRL), 25 ng/ml FGF-4 (Peprotech, New Jersey, USA), and 1 μg/ml heparin (Sigma-Aldrich, Missouri, USA) at 37°C in a humidified atmosphere of 5% CO2. For overexpression of human PRL-1 in Naïve PD-MSCs, a human PRL-1 plasmid containing the CMV6-AC vector (Origene, Inc., Rockville, MD, USA) was transfected into Naïve PD-MSCs using the 4D AMAXA NucleofectorTM system (Lonza, Basel, Switzerland) according to previous reports [17]. After harvest, PD-MSCs were labeled with a PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich). PD-MSCs (i.e., Naïve) and PD-MSCsPRL-1(i.e., PRL-1) at doses of 5×105 cells were injected intravenously via the tail vein in the transplanted animal group.
Construction of the OVX rat model and transplantation of stem cells
Female 7-week-old Sprague-Dawley rats (Orient Bio, Inc., Seongnam-si, Republic of Korea) were maintained in an air-conditioned animal facility. The rats were housed in groups of two rats per plastic cage with corn-cob bedding and were provided ad libitum access to standard commercial food and tap water. The temperature was 21°C, and a 12 h/12 h light-dark cycle was employed. The experimental procedures for the animal modeling and experiments were approved by the Institutional Animal Care and Use Committee of CHA University, Seongnam-si, Republic of Korea (IACUC-190007). The ovariectomy was performed by surgically removing one of the ovaries under general anesthesia with avertin (2,2,2-tribromoethanol, Sigma-Aldrich). Each rat was anesthetized through abdominal injection and avertin before the operation. Each group, including the normal, NTx and Tx groups, consisted of 5 rats. One week after OVX modeling, Naïve and PRL-1 (5x105 cells, 10–13 passages) labeled with a PKH67-linked kit (Sigma-Aldrich) were injected intravenously through the tail vein. All rats were sacrificed after 1, 3, and 5 weeks to harvest their ovarian tissues and blood samples.
RNA isolation and quantitative real-time polymerase chain reaction
Total RNA isolated from rat ovarian tissues using TRIzol reagent (Ambion, Thermo Fisher Scientific) according to the manufacturing’s protocol. Total RNA concentration was measured using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, USA). Total RNA was reverse transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen). The PCR conditions for the synthesis of cDNA were as follows: 5 min at 65°C, 1 min at 4°C, 60 min at 50°C, and 15 min at 72°C. The cDNA was used for qRT-PCR analysis. It was performed with SYBR Ex Taq (Roche, Basel, Switzerland). The cDNA was subsequently amplified by PCR under the following conditions: 5 seconds at 95°C and 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The sequences of the qRT-PCR primers are listed in Table S1. rGAPDH was used as an internal control for normalization, and each sample was analyzed in triplicate.
Ovarian explant ex vivo culture
For ovarian tissue cultivation, Matrigel (Corning, New York, USA) was added to a 24-well culture plate for 3 h. Then, each ovary of 7-week-old female rats was cut and rinsed with saline and DPBS containing 1% penicillin. For analysis of PRL-1 gene function in ovarian tissues, PD-MSCs treated with siRNA-PRL-1 at 50 nM (1x105 cells per ovary; Invitrogen) were directly injected into whole ovarian tissues after ovarian cultivation. After 48 h, the supernatant and ovarian tissues were collected for analysis. Next, to analyze the paracrine effect of PRL-1, an indirect cocultivation system was established using an insert system (Falcon). Naïve or PRL-1 cells were cocultured with the di-sectioned ovaries on an 8 μm pore size insert (SPL) and seeded at a density of 1x104 cells per insert with medium. For inhibition of PDGF, imatinib mesylate (1 μM; Sigma-Aldrich) was used to inhibit the expression of PDGF, and PDGF recombinant (10 ng/ml; PeproTech) was used to induce the expression of PDGF on the insert with medium. After 24 h and 48 h, the supernatant and di-sectioned ovaries were collected, and the samples were analyzed.
Nuclear fraction
Rat ovarian tissues from each group were measured at 20 mg in a 1.5 ml tube and diluted in 100 μl of CER I solution (NE-PERTM Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific) with phosphatase inhibitor. Tissues were vortexed and incubated on ice at 10 min. Five microliters of CERII solution (NE-PERTM Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific) was diluted in tubes. After vortexing, the samples were placed on ice for 1 min and centrifuged (~16000×g) for 5 min. After centrifugation, the supernatant (cytoplasmic) was transferred to clean prechilled tubes. Pellets were suspended in 50 μl of NER solution (NE-PERTM Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific). The samples were on ice and vortexed for 15 sec every 10 min for a total of 40 min. Then, the samples were centrifuged at maximum speed (~16000 g) in a microcentrifuge for 10 min. The supernatant (nuclear extract) fraction was transferred to clean prechilled tubes.
Protein isolation and western blotting
Rat ovarian tissues from each group were homogenized and lysed on ice with RIPA buffer (Sigma-Aldrich) containing protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (genDEPOT, Texas, USA). Equal concentrations of protein extracts were separated using 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, California, USA) using a Transfer Turbo system (Bio-Rad Laboratories). Membranes were blocked in blocking buffer (5% BSA) at room temperature for 1 h. Next, the membrane was incubated with primary antibody (1:1000) in 2% BSA at 4°C overnight. The following antibodies were mixed with 2% BSA and incubated at 4°C: rabbit anti-PDGF receptor β (28E1; 3169S, Cell Signaling Technology, Danvers, MA, USA) diluted 1:1000, mouse anti-PDGFR-α (C-9; sc-398206, Santa Cruz Biotechnology, Dallas, Texas, USA) diluted 1:100, mouse anti-HIF-1α (28b; sc-13515, Santa Cruz Biotechnology) diluted 1:5000, rabbit anti-CD105 (endoglin; bs-4609R; Bioss Antibodies, Woburn, MA, USA) diluted 1:1000, rabbit-anti VEGF receptor 2 (D5B1; 9698S, Cell Signaling Technology) diluted 1:1000, rabbit anti-total mTOR (7C10; 2983S, Cell Signaling Technology) diluted 1:1000, rabbit anti-mTOR (phospho S2448; ab109268, Abcam; Cambridge; MA; USA) diluted 1:1000, rabbit anti-LC3B (2775S, Cell Signaling Technology) diluted 1:1000, mouse anti-Erg-1/2/3 (D-3; sc-271048, Santa Cruz Biotechnology) diluted 1:1000, mouse anti-Nobox (D-3; sc-390016, Santa Cruz Biotechnology) diluted 1:1000, rabbit anti-BMP15 (MBS2516631, Mybiosource, San Diego, Ca, USA) diluted 1:1000, and rabbit anti-EGF receptor (2232S, Cell Signaling Technology) diluted 1:1000. After incubation, the membranes were washed with 1X Tris-buffered saline-Tween 20 (TBS-T) and then incubated with secondary antibody (1:10000) according to the manufacturer’s instructions at room temperature for 1 h. After the membranes were washed, they were treated with a Clarity Western ECL kit (Bio-Rad Laboratories) at room temperature for 5 min. The protein bands were detected by a ChemiDoc XRS+ imaging system (Bio-Rad Laboratories). The bands were analyzed by the ImageJ program (Wayne Rasband, Bethesda, Maryland). The fold change value of intensity is a comparative value of gene expression.
ELISA
All blood samples were collected from the aortas of rats in the normal, NTx and Tx groups. Individual serum samples were separated from whole blood by using a blood collection tube. (Vacutainer; BD Biosciences, San Jose, CA, USA). All blood serum was stored at −80°C, and estrogen (Biovision, Milpitas, CA, USA), AMH (Elabscience Biotechnology, MA, USA), FSH (Abnova, Taipei, Taiwan), and active caspase-3 (Mybiosource, San Diego, CA, USA) activity in serum was analyzed by ELISA kits following the manufacturer’s instructions. In brief, an equal volume of sample was added to the specific antibody-coated plates. Next, specific horseradish peroxidase (HRP)-conjugates were added to each well and incubated at 37°C. After the substrates had been added and incubated in the dark for substrate development, the antibody activity was analyzed by using a microplate reader (BioTek, Winooski, VT, USA).
H&E staining for follicle counting
Ovarian tissues were fixed with 10% neutral buffered formalin (BBC, Washington, USA), embedded in paraffin, and serially sectioned into 4 μm ovaries. Sectioned ovarian tissues were deparaffinized in a 60°C dry oven and by xylene and ethanol. Deparaffinized tissues were washed under tap water. The slides were dipped in Harris hematoxylin (Leica Biosystems, Wetzlar, Germany) for 7 min, dipped in 0.1% HCl for 2 seconds and counterstained with alcoholic eosin Y solution (Sigma-Aldrich). The stained slides were scanned for whole ovaries by 3D HISTECH (The Digital Pathology Company, Budapest, Hungary). The follicles were counted every 100 μm in serially sectioned slides and were defined as the total follicles, including primordial, primary, secondary, and preovulatory follicles, and the antral follicles according to previous reports [18]
Immunohistochemistry staining
Sectioned ovarian tissues were deparaffinized in a 60°C dry oven and by xylene and ethanol. Deparaffinized tissues were subjected to antigen retrieval by EDTA (eLbio, Seongnam-si, Republic of Korea) reaction and slowly cooled with water. The ovarian tissues were washed with distilled water (D. W) and treated with peroxide blocking solution containing 3% H2O2 in methanol for 10 min. Next, the ovarian tissues were washed with D.W and treated with primary antibodies with diluent buffer (Dako) at 4 °C overnight. The rabbit anti-PDGF receptor β antibody (28E1; 3169S, Cell Signaling Technology) was diluted 1:250. After removal of the unbound primary antibody, the tissues in slides were incubated with Dako Real EnVision HRP Rabbit/Mouse secondary antibody (Dako, California, USA) at room temperature for 1 h. The slides were incubated with DAB and counterstained with hematoxylin (Dako). After reaction, slides were rinsed by tap water. Slides were dehydrated by ethanol and xylene. Tissues were analyzed by the 3D HISTECT program (The Digital Pathology Company).
Immunofluorescence staining
Frozen ovarian section blocks were sectioned at 6 μm thickness and fixed with methanol for 10 min. After air drying, ovarian tissues were washed with 1X phosphate-buffered saline (PBS) at room temperature 3 times for 5 min each. Then, 1x PBS at the tissue edge was removed and placed in a humidified chamber. Tissues were treated with blocking solution (Dako) at room temperature for 1 h and treated with primary antibodies against each target gene at 4°C overnight. The following antibodies were mixed with antibody diluent buffer (Dako) and used: goat anti-PECAM-1 (CD31; sc-1506, Santa Cruz Biotechnology) diluted 1:250, rabbit anti-LC3B (2775S, Cell Signaling Technology) diluted 1:200, and rabbit anti-phospho-Src (Tyr527, 2105S, Cell Signaling Technology) diluted 1:200. Next, all tissues were incubated at room temperature for 1 h. The tissues were washed with 1X PBS at room temperature 3 times for 5 min and then treated with secondary antibody (1:250) at room temperature for 1 h. The cells were washed with 1X PBS at room temperature for 5 min 3 times. Then, the tissues were mounted with mounting medium with DAPI (Vectashield, Burlingame, CA, USA). The prepared slides were observed by fluorescence microscopy (Zeiss LSM 780, Oberkochen, Germany) at 400x and 630x magnification. All parts of each slide were observed, and representative images were captured.
HUVEC permeability dextran assay for vascular function
HUVECs were cultured with ECM medium (Science Cell, California, USA) at 37°C in a 5% CO2 incubator. Matrigel was coated on an insert with a 0.4 μm pore size (Falcon, New York, USA). After 3 h, HUVECs at a dose of 3x105 cells were seeded on an insert with a 0.4 μm pore size with medium. After 24 h, cells were treated with 100 μg/ml 5-FU. After treatment for 48 h, the medium was changed, and Naïve and PRL-1-overexpressing cells were seeded in 24-well plates (Falcon). After coculture for 24 h, inserts were transferred to new wells with medium, and dextran (Sigma-Aldrich) was added to the insert. After 20 min, medium on a 24-well plate was loaded in a 96-well assay black plate (Costar, Washington, USA), and the intensity was read with a Tecan assay.
Statistical analysis
All experiments were conducted in duplicate or triplicate. The results are presented as the mean ±standard error. Student’s t-test was used to analyze the groupwise comparisons. GraphPad Prism 5.0 (GraphPad Software, Inc., CA, USA) was used to conduct statistical analysis using one-way ANOVA followed by Tukey’s multiple comparisons test. P-values less than 0.05 were considered significant.