Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC 1500072) of the CHA laboratory animal research center at Sampyeong-dong in Gyeonggi, Korea. Female Sprague-Dawley rats (Orient Corporation, Seongnam, Gyeonggi, Korea) that were 8-weeks-old were used in this study. All rats were housed at two per pathogen-free cage at room temperature (21℃) with 12 hour light-dark cycle, given ad libitum access to water and standard commercial food.
Isolation and culture of chorionic-plate mesenchymal stem cells
The collection of human placenta and their use were approved by the Institutional Review Board (IRB) of CHA General Hospital, Seoul, Korea (IRB07-18). Placentas were collected from women who delivered at term (38±2 gestational weeks). Placenta-derived mesenchymal stem cells were isolated from normal chorionic plate of term placentas, as previously described [20]. PD-MSCs were cultured in DMEM/F12 medium supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA, USA), penicillin (100 U/ml; Thermo Fisher Scientific), streptomycin (100 µg/ml; Thermo Fisher Scientific), fibroblast growth factors 4 (25 ng/ml; Peprotech, Rocky Hill, NJ), and 1 µg /ml heparin (Sigma-Aldrich, St. Louis, MO, USA) at 37℃ under 5% CO2.
Generation of ovarian failure model by ovariectomy and PD-MSC transplantation into a rat model
Following acclimatization, the rats were randomly divided into three groups. The NTx group, ovariectomized (OVX) rats (n=25); the DTx group, directly PD-MSCs transplanted OVX-rats through ovary (n=45); the TTx group, indirectly PD-MSCs transplanted OVX-rats through tail vein injection (n=45). Ovariectomy was performed in rats of all groups to remove one of ovaries. All rats were anesthetized by intraperitoneal injection with 250 mg/kg Avertin (Sigma-Aldrich). The surgical site (pelvic area of the back) was disinfected with ethanol, and then, through the skin and muscle incision of 1~2 cm, one ovary was accessed and removed by excision. After removal of the ovary, the incision was sutured and was disinfected with povidone-iodine (Sigma-Aldrich). One week after the ovariectomy, PD-MSCs were stained using a PKH67 Fluorescent Cell Linker Kit (Sigma-Aldrich) and injected through the remaining ovary (DTx; 1 x 105 cells) or tail vein (TTx; 5 x 105 cells). Non-transplanted rats were injected with culture medium. Blood samples were collected weekly and measured for plasma E2 using an Estradiol DSL-4400 Radioimmunoassay Kit (Diagnostic System Laboratories, Inc, Webster, TX, USA) before sacrifice. Rats were sacrificed and ovary tissues were harvested at 1, 2, 3 and 5 weeks.
Ex vivo culturing of the ovary
PD-MSCs were plated in 24-well culture plates at density of 1x104 cells per well, cultured in modified-culture medium [α-MEM (Thermo Fisher Scientific) containing 2% FBS (Thermo Fisher Scientific), 1X penicillin/streptomycin (Thermo Fisher Scientific), 25 ng/ml FGF4 (Peprotech), and 1 µg /ml heparin (Sigma-Aldrich)] at 37℃ under condition of 5% CO2. After 24 hours, the inserts were pre-coated with matrigel (BD Biosciences, San Jose, CA, USA) for 3 hours and fitted on each well of 24-well plate containing PD-MSCs or only culture medium. The ovary tissues isolated from the normal rats were cut into half size using a sterile scalpel blade (JEUNGDO Bio and Plant Co., Ltd, Seoul, Korea) and placed in the upper chamber of the matrigel-coated insert. Co-culture plates were incubated in 37℃ incubator at 5% CO2 for 24, 48 and 72 hours.
Hormone ELISA assays
Blood samples were collected from rats of NTx, DTx, and TTx groups via the retro-orbital technique. Serum was separated from whole blood by using a serum separating tube (SST; BD Biosciences). Both the serum samples and the supernatants from the ex-vivo cultures were analyzed using ELISA kits for the anti-Mullerian hormone (AMH; Elabscience, Beijing, China), and the follicle-stimulating hormone (FSH, Abbexa, Cambridge, UK), according to the manufacturer’s instructions. Briefly, an equal volume of a given sample was added to the specific-antibody labeled plates. Then, specific HRP-conjugates were added to each well, and incubated. Next, the substrates were added to each well and incubated in dark. After the substrate development, the activity of the antibodies was analyzed using a microplate reader (BioTek, Winooski, VT). Concentration of Estradiol (E2) was measured by a chemiluminescence immunoassay (CLIA) using UniCel-DxI800 auto immunoassay analyzer (Beckman-Coulter, Brea, CA, USA).
Genomic DNA isolation
Genomic DNA (gDNA) was extracted from the rat ovaries by treatment with proteinase K (QIAGEN, Valencia, CA, USA) and phenol/chloroform (Sigma-Aldrich). This involved the ovary tissues of rats ground in LN2, followed by tissue powders digested in the digestion buffer [100 mM Tris pH 8.0 (Abelbio, Jungnang, Seoul, Korea) buffer containing 5 mM EDTA (Bioneer, Daedeok, Dajeon, Korea), 0.2% sodium dodecyl sulfate (SDS; Bioneer), 200 mM sodium chloride (Bioneer), and 0.5 mg/ml proteinase K (Dako, Cambridge, U.K)] at 55℃ and incubation overnight. The supernatants containing gDNA were extracted by using phenol/chloroform (1:1, Sigma-Aldrich), and damped down by isoamyl alcohol (Sigma-Aldrich) and 0.3 M sodium acetate (Bioneer) at -20℃ overnight. Next, the pellet of gDNA was washed with 70% cold ethanol and was eluted using Tris-EDTA buffer. The gDNA for each sample were analyzed using 1% agarose gel electrophoresis.
RNA isolation and quantitative real-time polymerase chain reaction analysis
Total RNA was homogenized and extracted from rat ovary using a Trizol reagent (Invitrogen Thermo Fisher, Camarillo, CA USA). Total RNA was reversely transcribed into cDNA using Superscript III RNase H reverse transcriptase (Invitrogen) and according to the manufacturer’s protocols. Briefly, first cDNA transcription of total RNA (500 ng) was performed with oligo dT (Invitrogen), and dNTP mix (Invitrogen) at 65℃ for 5 minutes followed by second cDNA synthesis performed at 50℃ for an hour and at 72℃ for 15 minutes with DTT (Invitrogen), RNase out (Invitrogen), Superscript III (Invitrogen), RNase H (Invitrogen) and reverse transcriptase (Invitrogen). cDNA and gDNA were amplified with specifically designed primers (Table S1) and detected using A SYBR Green master mix (Roche Diagnostics, Basel, Switzerland) and ExicyclerTM 96 PCR system (Bioneer). The cDNA amplification conditions for qRT-PCR were denaturation at 95℃ for 5 minutes followed by 40 cycles of 95℃ for 5 seconds, and 59℃ for 30 seconds. All experiments were performed in triplicate. The expression levels were calculated using the Ct method after normalization to mRNA levels of GAPDH as an internal control.
Western blots
Rat ovary tissues harvested from the animal experiments or the ex-vivo cultures were ground, sonicated, and lysed in protein lysis buffer [RIPA buffer (Sigma-Aldrich) containing a protease inhibitor cocktail (Roche) and phosphatase inhibitors (A.G. Scientific, San Diego, CA, USA)]. Total protein extracts (45 µg) were separated on 8%~15% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA). The membranes were blocked with 8% skim milk or 5% BSA (Amresco, Solon, OH, USA) for 1 hour at room temperature (RT), and then incubated with primary antibodies (1:1000) at 4℃ overnight, followed by secondary antibodies (1:20,000) for 1 hour at RT using an orbital shaker. The expression was detected with an ECL Advanced Western Blot Detection kit (Amersham, Marlborough, MA, USA) and by ChemiDoc™ XRS+ System (Bio-Rad). Antibodies used in this study included anti-rabbit-Nanos3 (Abcam, Cambridge, UK), anti-mouse-Nobox (Santa Cruz Biotechnology, Dallas, TX, USA), anti-goat-LHX8 (Santa Cruz), anti-rabbit-p-Akt (Cell Signaling, Danvers, MA, USA), anti-rabbit-PI3K (Cell Signaling), anti-rabbit-p-GSK3beta (Cell Signaling), anti-rabbit-p-FOXO3alpha (Cell Signaling), anti-rabbit-caspase9 (Abcam), anti-rabbit-caspase3 (BD Biosciences), anti-rabbit-GAPDH (Invitrogen), HRP-conjugated goat-anti-mouse IgG (Bio-Rad), HRP-conjugated donkey-anti-goat IgG (Sigma-Aldrich), anti-mouse and HRP-conjugated donkey-anti-rabbit IgG (Amersham). All experiments were performed in triplicate. Intensity of each band was quantified by Image J software (NIH, Bethesda, MD, USA)
Enzyme-linked immunosorbent assay (ELISA)
Stem cell factor (SCF) and caspase-3 activities in ovarian tissue lysates and supernatants of ex-vivo culture were measured with the SCF ELISA (R&D Systems, Minneapolis, MN, USA) and Caspase-3 ELISA kits (Promega, Gangseo, Seoul, Korea), respectively, and according to the manufacturer’s protocols. Also, cell necrosis was assessed by the release of lactate dehydrogenase (LDH) into the supernatants of the co-culture system. LDH activity was measured using a CytoTox 96 assay system (Promega) following the manufacturer’s protocol. All experiments were performed in triplicate.
Hematoxylin and eosin (H&E) staining
To evaluate the number of follicles in rat ovary after ovariectomy and PD-MSC transplantation, H&E staining was performed. Rat ovary samples were processed in 10% formalin (Millipore, Billerica, MA, USA). Five-micron-thick paraffin sections at were stained with H&E, and visualized by inverted light microscope. The total number of follicles less than 100 µm in diameter was counted in at least ten selected non-overlapping fields.
Immunostaining
To analyze the expression and localization of LHX8 and Lin-28 in rat ovary following the transplantation of PD-MSCs, ovary samples were embedded in OCT compound (Fisher Scientific, Pittsburgh, PA, USA). Five-micron-thick cryostat sections were fixed in cold methanol for 10 min and permeabilized with proteinase K (Dako) for 5 min at RT. The sections were blocked by protein block serum-free buffer (Dako) at RT for 30 minutes, incubated first with anti-goat-LHX8 (1:100 dilutions, Santa Cruz) at 4℃ overnight and next with anti-rabbit-Lin-28 (1:100 dilution, Abcam) in dark at RT for 2 hours. The mixture of secondary antibodies including Alexa Fluor 488 chicken anti-goat IgG (1:200 dilutions, Invitrogen) and Alexa Fluor 594 goat anti-rabbit IgG (1:200 dilution, Invitrogen) were incubated in antibody diluent (Dako) at RT for 1 hour followed by nuclei staining with 4’ ,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA, USA). The stained coverslips were mounted using mounting solution to avoid light loss. Images were visualized using an Olympus confocal microscope (x100 magnification) (Olympus, Tokyo, Japan https://www.olympus-global.com).
Statistical analysis
Data are represented as means and standard error (±SE). Significance values were calculated by Student’s t-test and an ANOVA on SAS software (SAS Institute, Cary, NC, USA) and p-value of <0.05 was used to determine significance (labeled as * or #).