Overexpression of Oct4 in porcine ovarian stem/stromal cells enhances differentiation of oocyte-like cells in vitro and ovarian follicular formation in vivo

All chemicals and media were purchased from Sigma (St. Louis, MO, USA) and Gibco (Life Technologies, Burlington, ON, Canada), respectively, unless otherwise specified. Media employed for washing was Dulbecco’s phosphate buffered saline (D-PBS) supplemented with 1 mg/ml poly vinyl alcohol (PVA), 1 % penicillin-streptomycin (10,000 IU and 10,000 μg/ml, respectively, Pen-Strep). Advanced Dulbecco’s modified Eagle’s medium (A-DMEM) supplemented with 10 % fetal bovine serum (FBS), 2 mM glutamine and 1 % Pen-Strep was used for cell culture. The pH and osmolality of all media were adjusted to 7.2 and 285 ± 5 mOsm/L, respectively.

All experiments were authorized by the Animal Center for Biomedical Experimentation at Gyeongsang National University. The two types of cells [i.e., OvSCs and adult fibroblasts (AFs)] were established from sexually mature 6-month-old female pigs. To isolate OvSCs, ovarian cortex was separated using fine forceps and blade under a microscope, minced into small pieces and then digested by incubating in DPBS containing 1 mg/ml collagenase type I for 1 h. Isolated cells were passed through 100 and 40 μm cell strainer (BD Falcon, MA, USA) sequentially to harvest single cell suspension and washed twice with D-PBS by centrifugation at 500 × g for 5 min. AFs were isolated from ear skin tissues. All the cells were cultured in A-DMEM supplemented with 10 % FBS at 38.5 °C in humidified atmosphere of 5 % CO₂ in air. Once confluence reached, ovarian stem/stromal cells (OvSCs) were trypsinized using 0.25 % trypsin-ethylenediaminetetraacetic acid (EDTA) solution and pelleted by centrifugation at 500 × g for 5 min. Cells were replated and passaged 4 times for further experiments.

Oct4 transfection was followed as per the recommended protocol (Allele biotechnology, CA, USA) with minor modifications. Briefly, a total of 1 × 10⁵ OvSCs were transfected with 20 MOI hOct3/4-RFP (O^R) (ABP-SC-LVIOCT3R, Allele) (Oct4-OvSCs) and polybrene (final concentration, 4 μg/ml) in 2 ml A-DMEM supplemented with 10 % FBS by incubating at 38.5 °C in a humidified atmosphere of 5 % CO₂ in air. Media was changed 10 h after transfection. OvSCs were also transfected by GFP as a lentiviral infection control. GFP construct was provided by hOct3/4-RFP (O^R) kit and the transfection method is same as hOct3/4-RFP (O^R) transfection.

To detect AP activity, the OvSCs attached to the plastic culture surface were fixed with 3.7 % formaldehyde for 1 h. and washed three times with Tris-HCl buffer [100 mM Tris-HCl (Trizma® hydrochloride), 50 mM NaCl, 50 mM MgCl2 · 6H2O and 0.1 % Tween-20]. The cells were stained with AP chromogen kit (BCIP/NBT) (Promega, WI, USA) for 30 min at room temperature. After being washed twice with deionized water, the cells were evaluated for positive activity under a microscope. AFs were used as a negative control.

The cells at passage 4 with approximately 80 % confluence were analyzed for the stage of cell cycle by fluorescence-activated cell sorting (FACS, Becton Dickinson, NJ, USA). Briefly, after fixation with 70 % ethanol solution at 4 °C overnight, the cells were washed with D-PBS twice and stained with 10 μg/ml propidium iodide in the presence of 2 mg/ml RNase A (Qiagen, Hilden, Germany) for 1 h. at 4 °C. A total of 1 × 10⁴ cells were analyzed for the DNA content in triplicates by flow cytometer and the DNA content of each cells was categorized as G0/G1, S or G2/M stage of the cell cycle.

For analysis of cell proliferation, cells were seeded at 1 × 10³ cells/well in a 12-well tissue culture dish in A-DMEM supplemented with 10 % FBS for 14 days. Total cell number of each well was counted with a hematocytometer at every 2 days interval.

Cells were analyzed for the expression of CD markers using flow cytometer. The cells at ~80 % confluence were harvested with 0.25 % trypsin EDTA treatment and blocked with 3 % bovine serum albumin (BSA) solution for 1 h. at 4 °C. For evaluation of CD29 expression, cells were incubated in primary antibody (Mouse anti-pig CD29, 5 μg/ml, BD Pharmingen™, CA, USA) for 1 h. at 4 °C. The cells were washed twice using D-PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (FITC goat anti-mouse IgG, 5 μg/ml, BD Pharmingen™) for 1 h. at 4 °C. For analysis of CD44, 45 and 90, cells were incubated with FITC-conjugated anti-CD44, 90 (5 μg/ml, BD Pharmingen™) and CD45 (2 μg/ml, Sigma, MO, USA) for 1 h. at 4 °C. The standard was established by appropriate isotype-matched negative controls. FITC–labeled cells were analyzed after acquisition of 1 × 10⁵ events by FACS caliber and Cell Quest software (Becton Dickinson). All experiments were performed in triplicates in three independent experiments.

Cells were fixed with 3.7 % formaldehyde solution for 1 h. at room temperature. After being washed twice with D-PBS, the cells were permeabilized with 0.1 % triton X-100 for 30 min and blocked in D-PBS supplemented with 1 % BSA solution, followed by overnight incubation at 4 °C with primary antibodies against Oct3/4 (Goat polyclonal, 1:200, Santa Cruz Biotechnology, Inc., CA, USA), Nanog (Goat polyclonal, 1:200, Santa Cruz Biotechnology), Sox2 (Rabbit polyclonal, 1:200, Santa Cruz), VASA (Rabbit polyclonal, 1: 100, Abcam, Cambridge, UK) and DAZL (Rabbit polyclonal, 1:100, Abcam) diluted in blocking solution. Slides were rinsed with D-PBS, and then incubated with FITC-conjugated donkey anti-goat IgG or goat anti-rabbit IgG (1:200, Jackson IR laboratories, Inc., PA, USA) for 45 min at 38.5 °C. Slides were then counterstaining with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature, mounted with Vectashield® (Vector Laboratories, Inc., CA, USA) and observed under a fluorescence microscope (Leica, Wetzlar, Germany).

The expressions of pluripotent transcriptional factors and lineage specific markers were analyzed by RT-PCR. Total RNA was extracted from the cells using RNeasy® mini kit (Qiagen, CA, USA) following the manufacturer’s protocol. cDNA synthesis was performed for 30 min at 55 °C using Omniscript reverse transcription Kit (Qiagen, CA, USA) using oligo-dT primer. PCR was performed using Maxime PCR Premix (iNtRON Biotechnology, Seongnam, Korea) supplemented with each primers and cDNA samples under the following conditions; pre-denaturation at 95 °C for 10 min, denaturation at 94 °C for 30 s, annealing at 56–64 °C for 30 s, elongation at 72 °C for 45 s and final extension at 72 °C for 10 min for 30 cycles using a Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). PCR products were loaded on 1 % agarose gel supplemented with 1 μg/ml ethidium bromide solution for electrophoresis. The results were analyzed by zoom browser EX5.7 software (Primetech, Korea). To quantify the gene expression levels, semi-quantitative RT-PCR was performed, using β-actin for normalization as a reference gene. The gene expression levels were presented as a fold induction with mean ± standard error (SE). The intensities of each band were measured by Gel viewer 1.5 (CURVEX Corp., DAIHAN, South Korea) and calculated against a base of β-actin intensity for relative quantitative analysis. Experiments were performed in triplicates in three independent experiments. The primers used for the study are presented in Table 1. Porcine Oct4 primer (Table 1) was not matched with any sequence in human Oct4 mRNA sequence (GenBank: NM_001285986.1) and it has only matched with porcine Oct4 mRNA Sequence (GenBank: KJ023671.1).

The expression of Oct3/4, Sox2 and Nanog proteins were evaluated by western blot analysis. Total protein was extracted with RIPA buffer (PIERCE, IL, USA) containing protease inhibitor, quantified using BCA protein assay kit (PIERCE, IL, USA). A total of 20 μg proteins was separated on SDS-PAGE for 60 min at 15 mA per plate and wet transferred electrically onto a nitrocellulose (NC) membrane (Millipore, Eschborn, Germany) for 45 min at 200 mA. NC membrane was blocked with 5 % skim milk in 0.1 % TBST (1 M Tris [pH 7.5], 5 N NaCl and 0.1 % Tween-20) for 1 h. at room temperature. After washing 3 times with 0.1 % TBST, the NC membrane was incubated with 1:200 dilution of Oct3/4, Sox2 and Nanog (Santa Cruz Biotechnology, CA, USA) and 1:1000 dilution of β-actin (Cell Signaling Technology, MA, USA) antibody in 0.1 % TBST – 5 % skim milk for overnight at 4 °C. After being washed 3 times with 0.1 % TBST, the NC membrane was incubated with 1:5,000 dilution of goat conjugate anti-rabbit IgG-HRP or donkey conjugate anti-goat IgG-HRP (Santa Cruz Biotechnology, CA, USA) in 0.1 % TBST – 5 % skim milk for 1 h. at room temperature. Immunoreactivity was detected by enhanced chemiluminescence (ECL; Supersignal® West Pico Chemiluminescent substrate, PIERCE), and then exposed to X-ray film (FUJI Photo Film Co., Ltd, Tokyo, Japan). To quantify the protein expression levels, semi-quantification was performed. The protein expression levels were normalized to β-actin protein levels and presented as a fold induction with mean ± S.E. Experiments were performed in triplicates in three independent experiments. Relative intensities of each band were calculated on the basis of β-actin intensity by Gel viewer 1.5 (CURVEX Corp., DAIHAN, South Korea).

OvSCs and Oct4-OvSCs were differentiated into OLCs following a previously published protocol [5] with minor modifications. Media for induction into OLCs consisted of DMEM and nutrient mixture F12 (DMEM:F12 = 1:1, v/v) supplemented with 5 % FBS, 5 % porcine follicular fluid, 0.23 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine and 0.1 mM β-mercaptoethanol. The cells were cultured in differentiation media for 45 days by changing the media twice a week.

Induced OLCs were confirmed by observing their morphology with increasing size to approximately 50 μm in diameter at day 15, 30 and 45 of differentiation. On day 45, OLCs were stained with 1 μg/ml DAPI and observed under a fluorescence microscope (Nikon, Tokyo, Japan). The expression of pluripotent transcriptional factor, Oct4, and germ cell specific markers such as growth differentiation factor 9b (GDF9b), C-Mos, Vasa, ZPC, Stella, c-kit and folliculogenesis marker, FSHR was analyzed by using RT-PCR. β-actin was used as a control gene. Further, the expressions of Oct3/4, Vasa and DAZL in OLCs were analyzed by immunocytochemistry. In vitro produced porcine mature oocytes were used for positive control for immunocytochemistry.

For evaluating the number of OLCs, cells were seeded at 1 × 10⁵ cells/well in a 24-well plate and differentiated into OLCs for 45 days. On day 45, the floating cells in each well were counted and measured the diameter of the cells using ocular micrometer (Nikon TE300, Japan). The OLCs were classified on their diameter into < 50 μm and > 50 μm in diameter, and if OLCs had zona pellucida, the measurements included its diameter. Experiments were performed in eight replicates in three independent experiments.

Before the cell transplantation, the cells were labeled with fluorescent lipophilic carbocyanine dye PKH26 according to the manufacturer’s instructions (Sigma, MO, USA). The labeled cells were then used for transplantation experiments.

Female BALB/C Nude mice (normal mice), aged 5–6 weeks, weighing approximately 18–20 g, were used in this study. To induce infertility, recipients were sterilized by intraperitoneal injection of busulphan (20 mg/kg, suspended in DMSO), followed by a booster injection after 2 weeks. After 2 weeks from the booster injection, the animals were divided into five groups: control (n = 5, not injected with PBS or cells), PBS injection (n = 5), AFs injection (n = 7), OvSCs injection (n = 10), and Oct4-OvSCs injection (n = 10). After being anesthetized with a combination of 1 μl/g (60 μg/g) Zolazepam/tiletamine (zoletil50, Verbac, France) and 0.5 μl/g Zylazine, mice were injected with 5 μl PBS alone or with 5 μl (1 × 10⁴ cells) of cell suspensions into ovarian cortex using glass pipettes with a 70 μm diameter. Injections of PBS or AFs were evaluated for negative controls in normal and infertile mice.

Sera collected from the mice at 4 weeks after PBS or cell injection was used to measure the estradiol 17β and FSH level using ELISA. Estradiol 17β and FSH were analyzed using enzyme immunoassay kit for estradiol (Oxford Biomedical Research, MI, USA) and FSH (Endocrine Technology, CA, USA) according to the manufacture’s protocol, respectively. Five mice were used for each group and all serum samples and standards were run in duplicate.

The mice were sacrificed at 4 weeks after PBS or cell injection, and ovaries were collected for histological assessment. The ovaries were fixed with 4 % paraformaldehyde for overnight. After being washed with D-PBS 3 times each for 5 min, the ovaries were dehydrated overnight with 20 % sucrose. The dehydrated ovaries were embedded in optical cutting temperature (OCT) compounds (Tissue-Tek®, CE, USA) and sectioned into 8 μm thickness and mounted onto slides. The slides were stained with hematoxylin and eosin (H&E) staining or 1 μg/ml DAPI for 5 min. Images were observed using optical microscope (Nikon TE300, Japan) or fluorescence microscope (Leica CTR600, Switzerland).

For immunohistochemistry, the rabbit specific horseradish peroxidase-diaminobenzidine (HRP-DAB) detection immunohistochemical kit (Abcam) was used. Briefly, sections were incubated with a hydrogen peroxide block solution for 10 min, followed by treating protein block solution for 30 min. After being washed by D-PBS, sections were incubated with the primary antibody, anti-estrogen receptor alpha (rabbit polyclonal, 1:100, Abcam) at 4 °C overnight. Biotinylated goat anti rabbit IgG (H + L) was treated to section for 10 min as a secondary antibody. Visualization was detected using HRP-DAB detection IHC kit according to the manufacturer’s instructions. After counterstaining with hematoxylin, the sections were mounted and observed under the microscope.

In primary culture, the OvSCs were grown as heterogeneous populations with elongated, sphere-like or spindle-like adherent features. However, from passage 2–3, the cells became a homogeneous population with a typical fibroblast-like morphology (Fig. 1Aa). After Oct4 transfection of OvSCs, Oct4-OvSCs were also grown into homogeneous populations with a typical fibroblast-like morphology at passage 4 (Fig. 1Ad). OvSCs were transfected with lentiviral vector which expressed GFP as an infection control, and the infection rate in Oct4-OvSCs was found to be 66.7 ± 0.7 %, while non-transfected cells were not detected for GFP (Fig. 1e). At passage 4, OvSCs (Fig. 1Ab and Ac) and Oct-OvSCs (Fig. 1Af and Ag) were positive for AP activity and more population were observed for AP positivity in Oct4-OvSCs compared to OvSCs. These results indicated the existence of stem cells in ovarian cortex and proved the potential stemness in OvSCs transfected with exogenous Oct4. AFs were observed to be negative for AP activity (Fig. 1Ah and Ai).

The expression of mesenchymal cell surface specific markers, such as CD29, CD44 and CD90 were analyzed in OvSCs and Oct4-OvSCs using flow cytometer at passage 4 (Fig. 1b). The results were presented as percentage mean positive cells of surface markers in analyzed cell population. The percentage of cells that expressed CD44 was significantly (P <0.05) greater in OvSCs than that in Oct4-OvSCs (98.7 ± 0.7 % vs. 94.7 ± 0.3 %). CD29 and CD90 were highly expressed in both OvSCs and Oct4-OvSCs with no significant differences. However, the hematopoietic cell lineage marker, CD45, was not detected in OvSCs and Oct4-OvSCs (Fig. 1b).

Flow cytometer analysis showed that the population of G0/G1 phase of the cell cycle in Oct4-OvSCs was significantly (P <0.05) lower than OvSCs (43.8 ± 0.5 % vs. 51.5 ± 0.3 %) (Fig. 1c). In contrast, the population of S phase of the cell cycle in Oct4-OvSCs was significantly (P <0.05) higher than OvSCs (30.7 ± 0.7 % vs. 23.6 ± 0.8 %). These results indicated that the Oct4 transfection resulted in enhanced cell proliferation. The population of cells at G2/M phase in Oct4 transfected cells and OvSCs was observed to be 25.5 ± 0.6 and 24.9 ± 0.7 respectively.

Further, to confirm the cell proliferation ability, OvSCs and Oct4-OvSCs were compared for growth kinetics at passage 4 (Fig. 1d). Growth pattern of these cells revealed a sigmoid curve, wherein proliferation of Oct4-OvSCs was significantly (P <0.05) more rapid than OvSCs from day 6 to 14. There was no significant difference in cell proliferation between OvSCs and Oct4-OvSCs at day 2 and 4 of culture.

The expression of pluripotent transcriptional factors, such as Oct3/4, Sox2 and Nanog, in OvSCs and Oct4-OvSCs was analyzed by RT-PCR and western blot analysis (Fig. 2). The exogenous Oct4, which was transfected using human Oct3/4 lentiviral particle, was expressed in Oct4-OvSCs, whereas in OvSCs, it was not expressed (Fig. 2a). The endogenous Oct4 was expressed in OvSCs and Oct4-OvSCs as revealed by RT-PCR (Fig. 2a). However, the intensity of endogenous Oct4 was significantly (P <0.05) higher in Oct4-OvSCs than OvSCs (Fig. 2c). The Sox2 was also expressed in OvSCs and Oct4-OvSCs, and there was no significant difference between these cells based on band intensities (Fig. 2a and c). Nanog was also expressed in OvSCs and Oct4-OvSCs, with significantly (P <0.05) higher level in OvSCs than Oct4-OvSCs (Fig. 2a and c). Protein levels of Oct3/4, Sox2 and Nanog were evaluated by western blot analysis. Oct3/4 protein was expressed only in Oct4-OvSCs, but not in OvSCs (Fig. 2b and d). Sox2 and Nanog were detected in OvSCs and Oct4-OvSCs, with no significant difference among these cells (Fig. 2b and d).

Before induction into OLCs, OvSCs and Oct4-OvSCs had a fibroblast-like morphology (Fig. 1Aa and Ac). Following the differentiation, the cells changed to a distinct sphere-like morphology during the next 15 to 30 days of culture in the differentiation media (OvSCs, Fig. 3Aa and Ab; Oct4-OvSCs, Fig. 3Ba and Bb). At day-45 of differentiation, the morphology of cells was changed to ‘big round-cells’, which had approximately 50 μm in diameter, so called oocyte-like cells (OLCs) (Fig. 3Ac and Bc), which characteristically possessed zona pellucida like structure. Further, nuclear staining by DAPI was observed in OLCs derived from OvSCs (Fig. 3Ad and Ae) and Oct4-OvSCs (Fig. 3Bd and Ae) under a fluorescent microscope. OLCs derived from Oct4-OvSCs expressed a cytoplasmic red fluorescence protein (Fig. 3Be).

The number and diameter of OLCs derived from Oct4-OvSCs and OvSCs was analyzed by counting the floating cells (Fig. 3c). At day-45 of differentiation, the floating cells were counted and diameter of the cells was measured. The number of floating cells, less than 50 μm in diameter, was similar between Oct4-OvSCs and OvSCs (800.0 ± 44.3 vs. 818.7 ± 38.9). However, the OLCs from Oct4-OvSCs were generated significantly (P <0.05) higher number of cells with > 50 μm in diameter than OvSCs (68.7 ± 1.7 vs. 51.4 ± 2.9).

To characterize the differentiated cells into OLCs from OvSCs and Oct4-OvSCs, the expression of transcription factor (Oct4), germ cell and meiosis specific markers (Vasa, GDF9b, C-mos, C-kit, Stella and ZPC), and folliculogenesis marker (FSHR) was evaluated at mRNA level using RT-PCR. The relative expression levels were quantified using band intensities (Fig. 4a and b). Ovarian tissues were used as positive control and they expressed all oocyte specific markers (Fig. 4a). Oct4, Vasa, GDF9b, C-mos, FSHR and C-kit were detected in OvSCs and Oct4-OvSCs at all time points during the differentiation that were examined (Fig. 4a). Overexpression of Oct4 was displayed with higher level of Oct4 at each point till day 45 of differentiation of Oct4-OvSCs than OvSCs based on band intensities. However, the expression level of Oct4 did not differ between day 0 and 45 of differentiation in both OvSCs and Oct4-OvSCs (Fig. 4b). The expression of Vasa was significantly (P <0.05) increased in Oct4-OvSCs after differentiation into OLCs, but not in OvSCs (Fig. 4b). Further, there was no significant difference in the expression of Vasa among OvSCs and Oct4-OvSCs on day 45 of differentiation. GDF9b, C-mos and FSHR were expressed significantly (P <0.05) higher in both OvSCs and Oct4-OvSCs on day 45 of differentiation (Fig. 4b), although differences between cell types were not significant. C-kit level was significantly (P <0.05) higher in Oct4-OvSCs than OvSCs on day 45 of differentiation, but there was no significant difference between day 0 and 45 of differentiation (Fig. 4b). The expression of Stella initiated on day 45 and 15 of differentiation in OvSCs and Oct4-OvSCs, respectively (Fig. 4a). However, there was no significant difference in relative mRNA level of Stella between OvSCs and Oct4-OvSCs on day 45 of differentiation (Fig. 4b). The expression of ZPC initiated on day 15 of differentiation in both OvSCs and Oct4-OvSCs, and its expression level was found to be significantly (P <0.05) higher in Oct4 OvSCs compared to OvSCs on day 45 of differentiation (Fig. 4a and b). OLCs derived from OvSCs and Oct4-OvSCs were further analyzed by immunocytochemistry to detect the expression of Oct3/4, Vasa and DAZL at protein level (Fig. 5). Oct3/4 was expressed in the nucleus of OLCs, whereas Vasa and DAZL were detected in the cytoplasm (Fig. 5a and b) and these expression patterns were similar to in vitro matured oocytes (Fig. 5c).

Control BALB/C nude mice ovary revealed intact follicles containing oocytes and were found to be progressing through folliculogenesis (Fig. 6Aa and Ba). Upon injecting busulphan twice at 2 week intervals resulted in a disruption of folliculogenesis and destruction of germ cells including oocytes in ovaries when observed at 2 and 6 weeks after the second injection (Fig. 6Ab and Ac). A total of 1 × 10⁴ cells were transplanted into ovaries of busulphan treated mice. At 4 weeks after OvSCs transplantation into sterile ovaries, primary follicles were observed in the ovaries (Fig. 6Af and Ag). Furthermore, the ovaries transplanted with Oct4-OvSCs showed not only primary follicles (Fig. 6Ai) but also developing follicles containing theca and granulosa cells (Fig. 6Ah and Ai). Under the fluorescence microscopy, Oct4-OvSCs labeled with PKH26 were observed in developing follicles (Fig. 6Bf and Bg). However, OvSCs and Oct4-OvSCs labeled with PKH26 were not located in primary follicles (Fig. 6Be and Bh). The labeled AFs did not induce any follicles in sterile ovaries (Fig. 6Ad and Bc). The labeled cells were not observed in PBS injected ovaries (Fig. 6Ae and Bd).

Estrogen receptor alpha (ERα) was expressed in theca cells and interstitial cells in normal ovaries that were not injected with busulphan (Fig. 7a). After injection of busulphan in mouse, ERα was expressed in all interstitial cells in sterile ovaries (Fig. 7b). AFs or PBS injected ovaries also expressed ERα in all interstitial cells in infertile mice (Fig. 7c and d). In OvSCs transplanted ovaries, ERα was not expressed in primary follicles containing granulosa cells, but expressed in interstitial cells (Fig. 7e). ERα was expressed in theca cells of developing follicles and interstitial cells, but not in primary follicles in Oct4-OvSCs transplanted ovaries (Fig. 7f and g).

To evaluate the concentration of estradiol and FSH in mouse serum, ELISA analysis was performed (Fig. 8a and b, respectively). Control infertile mice revealed significantly (P <0.05) lower estradiol concentration than normal female BALB/C Nude mice (26.0 ± 3.3 vs. 52.7 ± 6.1 pg/ml), and showed similar estradiol concentration to AFs or PBS injected infertile mice (28.5 ± 3.6 and 25.75 ± 5.4 pg/ml). However, OvSCs or Oct4-OvSCs transplantation resulted in higher estradiol concentration in infertile mice. The concentrations of estradiol in OvSCs injected mice (45.5 ± 3.3 pg/ml) were lower than normal and Oct4-OvSCs injected mice (56.5 ± 4.3 pg/ml), but there was no significant difference among these groups.

FSH concentration of control infertile mice was significantly (P <0.05) higher than normal mice (62.5 ± 13.2 vs. 5.1 ± 0.9 ng/ml). After injection of AFs or PBS into infertile mice, FSH concentration was still significantly (P <0.05) higher than normal mice (59.1 ± 12.0 and 61.6 ± 16.3 vs. 5.1 ± 0.9 ng/ml). However, FSH concentration decreased by transplantation of OvSCs or Oct4-OvSCs into infertile mice. OvSCs or Oct4-OvSCs injected mice had lower FSH concentration than control infertile mice (23.8 ± 5.4 and 21.8 ± 4.6 vs. 62.5 ± 13.2 ng/ml). The FSH concentration of OvSCs or Oct4-OvSCs was higher than normal mice, but there was no significant difference.

This study was approved by the Animal Center for Biomedical Experimentation at Gyeongsang National University via GNU-120404-M0006.

Not Applicable.

Not Applicable.