Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Lin Liu ( liulin@nankai.edu.cn ). This study did not generate new unique reagents. All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

The method of isolation of GCs was described previously (). Briefly, pregnant mare serum gonadotropin (PMSG, Millipore, 5 IU per mouse) was injected into the abdominal cavity of 6-week-old female OG2 mice 46 h prior to isolating GCs. The mice were humanely sacrificed and the ovaries dissected. To isolate GCs, an insulin syringe was used to puncture visible follicles on the surface of ovaries under a stereomicroscope to release GCs into culture medium. The maturing or mature follicles in principle do not contain the primordial oocytes. In addition, 40 μm Cell Strainer were used to wipe off oocytes if any. Then, the freshly isolated GCs were washed in PBS and cultured in S/L medium, and the medium was changed every 48 h. Cells were cultured at 37°C in 5% COincubator with humidified air.

ESC lines were established and characterized based on the method described (). Blastocysts were isolated from the uterine horns of pregnant females at Embryo (E) day 3.5 using a dissection microscope in HKSOM and plated onto mitomycin C-treated MEF cells served as feeders in KSR/DMEM added with PD0325901 and LIF (K/DL) medium and cultured for 7 days to form outgrowths. Emerging ICM outgrowths were directly picked into serum/LIF (S/L) medium on feeders to establish stable ESC lines. Established ESC lines were genotyped by Sry gene to determine the sex. Female ESC lines were maintained by dissociating cells with 0.25% TE (Invitrogen) every 2∼3 days and re-plated onto feeder cells. K/DL medium contains knockout DMEM (Invitrogen) supplemented with 20% Knockout serum replacement (KSR, Invitrogen), 1 mM L-glutamine, 1% nonessential amino acid stock, 50 units/ml penicillin and 50 μg/ml streptomycin (2A, Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), 1 μM PD0325901 (Miltenyi) and 1000 IU/ml mouse LIF (mLIF, Millipore). S/L medium (ESC culture medium) contains knockout DMEM supplemented with 15% FBS (ES quality, Hyclone), 1 mM L-glutamine, 1% nonessential amino acid stock, 50 units/ml penicillin and 50 μg/ml streptomycin, 0.1 mM β-mercaptoethanol, and 1000 IU/ml mLIF. ESCs were cultured at 37°C in 5% COincubator with humidified air.

MEFs were derived from E13.5 embryos isolated from ICR mice by caesarean section and washed in phosphate-buffered saline (PBS). Heads and visceral tissues were removed, and remaining tissue washed in PBS, then submerged in 0.25% trypsin-EDTA (0.25% TE, Invitrogen) and incubated at 37°C for 10 min. Tissue was pipetted repeatedly to aid dissociation, washed then dissociated cells added to MEF medium and plated (passage 0, P0). MEFs were genotyped by Sry gene to determine the Sex (Forward primer: CATCGGAGGGCTAAAGTGTC; Reserve primer: TCCAGTCTTGCCTGTATGTG). MEF medium contains DMEM (Invitrogen) supplemented with 10% Fetal Bovine Serum (FBS, Hyclone), 1 mM L-glutamine (Invitrogen), 1% nonessential amino acid stock (NEAA, Sigma), and 50 units/ml penicillin and 50 μg/ml streptomycin (2A, Invitrogen). Cells were cultured at 37°C in 5% CO 2 incubator with humidified air (Thermo Scientific USA).

Use of mice for this research was approved by the Nankai University Animal Care and Use Committee. All mice used in this study were taken care of and operated according to the relevant regulations. Mice at the age of 4-8 weeks old were healthy, housed and cared in individually ventilated cages (IVCs) on a standard 12 h light: 12 h dark cycle in the sterile Animal Facility at College of Life Sciences. Male and female Oct4-GFP (OG2) mice (C57BL6 X CBA, JAX stock #004654) that carry Oct4 distal promoter-driven GFP were purchased from Model Animal Research Center of Nanjing University. Nu/Nu mice (Female, #403), NOD-SCID mice (Female, #406), albino BALB/c mice (Female, #401), albino ICR mice (Male and female, #201) and albino Kunming (KM) mice (Female, #202) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

Method Details

Proliferation Curve of Granulosa Cells The primary cultured GCs for 7 days (P0) were dissociated with 0.25% TE, washed, centrifuged, and resuspended in S/L medium, followed by seeding at a density of 105 cells per well in 6 well plate in S/L medium added with 10 μM Y27632 (Rock inhibitor, Rocki, Selleck) for only 24 h to facilitate cell adherence. Next day, the culture (considered as P1) was changed into S/L medium without Y27632. At various days, the cultured GCs were dissociated into single cells by 0.25% TE, washed, centrifuged, resuspended in PBS, and counted by Hemocytometer.

Induction of gPSCs from Granulosa Cells Zhao et al., 2015 Zhao Y.

Zhao T.

Guan J.

Zhang X.

Fu Y.

Ye J.

Zhu J.

Meng G.

Ge J.

Yang S.

et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. Fu et al., 2018 Fu H.

Tian C.L.

Ye X.

Sheng X.

Wang H.

Liu Y.

Liu L. Dynamics of Telomere Rejuvenation during Chemical Induction to Pluripotent Stem Cells. 5 cells per well in 6 well plate in S/L medium added with 10 μM Y27632 for only 24 h or without Y27632 served as control. Next day (0 D of induction), the culture (considered as P1) was changed into the Stage 1 induction medium for CiPSC induction. GCs were freshly isolated from OG2 mice that carry Oct4 distal promoter-driven GFP. Induction to pluripotent stem cells (PSCs) by small chemicals was performed based on the method described (), with modifications (). The primary cultured GCs for 7 days (P0) were dissociated with 0.25% TE, washed, centrifuged, and resuspended in S/L medium, followed by seeding at a density of 10cells per well in 6 well plate in S/L medium added with 10 μM Y27632 for only 24 h or without Y27632 served as control. Next day (0 D of induction), the culture (considered as P1) was changed into the Stage 1 induction medium for CiPSC induction. On 0-12 D (The first part of Stage 1), the induction medium contained 100 ng/ml bFGF (PeproTech), 0.5 mM VPA (Sigma), 20 μM CHIR99021 (Selleck), 10 μM Repsox (Selleck), 5 μM Parnate (Sigma), 50 μM Forskolin (Selleck), 0.05 μM AM580 (Tocris) and 5 μM EPZ004777 (Selleck). During 12-16 D (The second part of Stage 1), concentrations of bFGF, CHIR99021, and Forskolin were reduced to 25 ng/ml, 10 μM, and 10 μM, respectively. On 16-28 D (Stage 2), the induction medium contains 25 ng/ml bFGF, 0.5 mM VPA, 10 μM CHIR99021, 10 μM Repsox, 5 μM Parnate, 10 μM Forskolin, 0.05 μM AM580, 0.05 μM DZNep (Selleck), 0.5 μM 5-aza-dC (Sigma), and 5 μM SGC0946 (Selleck). On 28 D (Stage 3), the culture was transferred into 2i/L medium. 2i/L medium contains a 1:1 mixture of DMEM/F12 (Invitrogen) supplemented with N2 and B27 (Invitrogen), 1 mM L-glutamine, 1% nonessential amino acid stock, 50 units/ml penicillin and 50 μg/ml streptomycin, 0.1 mM β-mercaptoethanol, 1000 IU/ml mLIF, 1 μM PD0325901 and 3 μM CHIR99021. After 8-12 days, Oct4 GFP-positive, GCs-derived PSCs (gPSC) primary colonies emerged and were then picked up for expansion by culture in S/L + 2i medium on feeders. In comparison experiments by treatment with or without crotonic sodium (CS, crotonic acid (Millipore), added with 1 N NaOH to adjust the pH = 7.0) during induction, 7 mM CS was added into induction medium during Stage 2 from 16 D to 28 D. Oct4 GFP-positive cells emerged about from 32 D-36 D, and clones can be picked up at 40 D. For FACS analysis of reprogramming efficiency, cells at 40 D of induction were collected and washed three times with PBS and FACS analysis performed using a Flow Cytometer (Aria III, BD Biosciences).

Induction of PGC-like Cells Hayashi and Saitou, 2013 Hayashi K.

Saitou M. Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells. 5 female ESCs/gPSCs on the wells of a 12-well plate coated with human plasma fibronectin (FN, 16.7 μg/ml, Millipore) in N2B27 medium containing activin A (20 ng/ml, PeproTech), bFGF (12 ng/ml), and 1% KSR for 2 days. The medium was changed every day. The PGCLCs were induced for 4 days under floating conditions by plating 3.0 × 103 EpiLCs in the wells of a low-cell-binding U-bottom 96-well Lipidure-Coat plate (Corning) in GK15 medium (GK15 medium contains GMEM (Invitrogen) added with 15% KSR, 1 mM sodium pyruvate, 1 mM L-glutamine, 1% nonessential amino acid stock, 50 units/ml penicillin and 50 μg/ml streptomycin, and 0.1 mM β-mercaptoethanol) in the presence of the cytokines BMP4 (500 ng/ml, PeproTech), 1000 IU/ml mLIF, SCF (100 ng/ml, PeproTech) and EGF (50 ng/ml, PeproTech). Integrin-β3- (CD61) and SSEA1-double positive ESCs/gPSCs-derived PGCLCs were sorted by flow cytometer. Dissociated cells were incubated with anti-integrin-β3 (CD61) antibody and anti-SSEA1 antibody conjugated with PE (Phycoerythrin, Red color) and Alexa Fluor 647, respectively. After being washed in PBS supplemented with 0.1% bovine serum albumin (BSA, Sigma), the cells were sorted and analyzed using a flow cytometer (Aria III; BD Biosciences). Induction of PGC-like cells (PGCLCs) from ESCs/gPSCs was based on the method described previously (). ESCs and gPSCs at passage 7-8 were transferred into feeder-free N2B27+t2iL medium (in 2iL medium, the concentration of PD0325901 was reduced from 1 μM to 0.4 μM) and maintained for 3-5 passages. EpiLCs were induced by plating 1.0 × 10female ESCs/gPSCs on the wells of a 12-well plate coated with human plasma fibronectin (FN, 16.7 μg/ml, Millipore) in N2B27 medium containing activin A (20 ng/ml, PeproTech), bFGF (12 ng/ml), and 1% KSR for 2 days. The medium was changed every day. The PGCLCs were induced for 4 days under floating conditions by plating 3.0 × 10EpiLCs in the wells of a low-cell-binding U-bottom 96-well Lipidure-Coat plate (Corning) in GK15 medium (GK15 medium contains GMEM (Invitrogen) added with 15% KSR, 1 mM sodium pyruvate, 1 mM L-glutamine, 1% nonessential amino acid stock, 50 units/ml penicillin and 50 μg/ml streptomycin, and 0.1 mM β-mercaptoethanol) in the presence of the cytokines BMP4 (500 ng/ml, PeproTech), 1000 IU/ml mLIF, SCF (100 ng/ml, PeproTech) and EGF (50 ng/ml, PeproTech). Integrin-β3- (CD61) and SSEA1-double positive ESCs/gPSCs-derived PGCLCs were sorted by flow cytometer. Dissociated cells were incubated with anti-integrin-β3 (CD61) antibody and anti-SSEA1 antibody conjugated with PE (Phycoerythrin, Red color) and Alexa Fluor 647, respectively. After being washed in PBS supplemented with 0.1% bovine serum albumin (BSA, Sigma), the cells were sorted and analyzed using a flow cytometer (Aria III; BD Biosciences).

Induction of Meiosis and Folliculogenesis Zeng et al., 2017 Zeng M.

Sheng X.

Keefe D.L.

Liu L. Reconstitution of ovarian function following transplantation of primordial germ cells. Induction of meiosis and folliculogenesis was achieved by aggregation of PGCLCs or PGCs served as a control with E12.5 gonadal somatic cells followed by transplantation of the aggregates into kidney capsules to generate reconstituted ovaries (rOvaries). To obtain E12.5 gonad somatic cells in vivo, female E12.5 gonads were collected from E12.5 embryos obtained by intercrosses of albino ICR mice. The mesonephros were surgically separated from the gonads using insulin syringe. Gonads were dissociated with 0.05% TE by incubation at 37°C for 10 min, washed with MEF medium, and collected by centrifugation. Large clumps of cells were removed using a cell strainer (BD Biosciences). Endogenous PGCs in the dissociated gonadal cells were removed or collected by magnetic cell sorting using anti-SSEA1 antibody conjugated with magnetic beads (Miltenyi, see below). The sorted gonadal somatic cells and FACS-sorted PGCLCs or PGCs from OG2 mice were plated in the wells of a low-cell-binding U-bottom 96-well Lipidure-Coat plate in MF10 medium (MF10 medium contains M199 (Sigma) added with 10% FBS, 1 mM L-glutamine, 1% nonessential amino acid stock, and 50 units/ml penicillin and 50 μg/ml streptomycin) with or without 50 μg/ml Vitamin C (Vc, Sigma) or/and 10 μM Rocki (). Each aggregate contained 20,000 E12.5 PGCs or PGCLCs and 100,000 gonadal somatic cells. For negative control (pseudo-rOvary), 100,000 E12.5 gonadal somatic cells only without PGCs were aggregated in MF10 medium supplemented with 50 μg/ml Vitamin C and 10 μM Rocki.

Magnetic Activated Cell Sorting Magnetic activated cell sorting (MACS) was performed according to the manufacturer’s instructions (Miltenyi). Briefly, dissociated gonadal cells were incubated with anti-SSEA1 antibody conjugated with magnetic beads. Cell suspensions were washed in PBS supplemented with 0.5% BSA and 2 mM EDTA and applied to an MS column (Miltenyi) to remove SSEA1-positive PGCs. Gonadal somatic cells were collected in the flow-through portions. To verify that SSEA1 negative cells do not contain PGCs, SSEA1/ CD61 double staining was used as markers to detect PGCs by FACS. More than 99.99% cells did not express SSEA1 and CD61, indicating the purity of embryonic gonadal somatic cells.

Kidney Capsule Transplantation Qing et al., 2008 Qing T.

Liu H.

Wei W.

Ye X.

Shen W.

Zhang D.

Song Z.

Yang W.

Ding M.

Deng H. Mature oocytes derived from purified mouse fetal germ cells. Zeng et al., 2017 Zeng M.

Sheng X.

Keefe D.L.

Liu L. Reconstitution of ovarian function following transplantation of primordial germ cells. Kidney capsule transplantation was performed based on the methods described (). Briefly, one or two aggregates were implanted in the “pocket” which was made between the kidney capsule and kidney tissue of a bilaterally ovariectomized recipient mouse. Transplantation procedure was completed in 5 min for each mouse. Meiosis and folliculogenesis were achieved in the rOvaries 28 days following transplantation of the aggregates.

In Vitro Maturation and In Vitro Fertilization Eppig et al., 2009 Eppig J.J.

O’Brien M.J.

Wigglesworth K.

Nicholson A.

Zhang W.

King B.A. Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring. The rOvaries were dissected from the recipient mouse kidney capsule, and fully-grown GV oocytes were collected under a microscope by pricking follicles using insulin syringe in in vitro maturation (IVM) medium. Oocytes were matured in vitro by culture in IVM medium for 17-18 h at 37°C (). IVM medium contains α-MEM (Invitrogen) added with 5% FBS, 0.24 mM sodium pyruvate, 1 IU/ml PMSG and 1.5 IU/ml human chorionic gonadotropin (hCG, Sigma). Oocytes at MII, determined by extrusion of the first polar body, were subjected to IVF. With this IVM method, 80% of maturation rate was routinely obtained for oocytes collected in vivo. AA medium for blastocyst transfer into the uterus of E2.5 pseudo-pregnant mice, and newborns were normally delivered on E19.5. Pups were identified initially by coat color. The contribution of E12.5 PGCs, ESCs or gPSCs to various tissues of pups was confirmed by GFP primer and by standard DNA microsatellite genotyping analysis using D8Mit94 and D12Mit136 primers ( For in vitro fertilization (IVF), spermatozoa were collected from the cauda epididymis of ICR males, capacitated by incubation for 2 h in HTF (Origio), and then incubated with the matured oocytes for 6 h. The zygotes were transferred into human G-1 plus medium (Vitrolife). Embryos that reached the 2-cell stage after 24 h culture were transferred into the oviducts of E0.5 pseudo pregnant mice or cultured in KSOMmedium for blastocyst transfer into the uterus of E2.5 pseudo-pregnant mice, and newborns were normally delivered on E19.5. Pups were identified initially by coat color. The contribution of E12.5 PGCs, ESCs or gPSCs to various tissues of pups was confirmed by GFP primer and by standard DNA microsatellite genotyping analysis using D8Mit94 and D12Mit136 primers ( Table S2 ).

Embryoid Body Formation Assay ESCs and gPSCs were removed off feeder cells twice based on their differences in the adherence to the bottom of dish. The ESCs or gPSCs were diluted to 4 × 104/ml. Every 30 μL was pipetted to form a hanging drop on the cover of the 100 mm dish. Embryoid body (EBs) formed on day 4 and were transferred into 6-well plates for adherent culture, and fixed for immunofluorescence staining using markers of three embryonic germ layers on day 15.

Teratoma Formation Assay 1 × 106 ESCs and gPSCs were injected subcutaneously into about 6-week-old female Nu/Nu mice. About 4 weeks after injection, the mice were humanely sacrificed, and the teratomas were excised, fixed in 4% paraformaldehyde, dehydrated in gradient ethanol, embedded in paraffin, and sectioned for histological examination by hematoxylin and eosin (H&E) staining.

Production of Chimeras and Genotyping Huang et al., 2008 Huang J.

Deng K.

Wu H.

Liu Z.

Chen Z.

Cao S.

Zhou L.

Ye X.

Keefe D.L.

Liu L. Efficient production of mice from embryonic stem cells injected into four- or eight-cell embryos by piezo micromanipulation. AA medium. Blastocysts were transferred into uterine horns of E2.5 surrogate mice. Pregnant females delivered pups naturally at about E19.5. Pups were identified initially by coat color. The contribution of ESCs or gPSCs to various tissues in chimeras was confirmed by standard DNA microsatellite genotyping analysis using D12Mit136 primers ( Approximately 10-15 ESCs or gPSCs at P8-12 were injected into 4-8-cell embryos of BALB/c mice as hosts using a Piezo injector as described (). Injected embryos were cultured overnight in KSOMmedium. Blastocysts were transferred into uterine horns of E2.5 surrogate mice. Pregnant females delivered pups naturally at about E19.5. Pups were identified initially by coat color. The contribution of ESCs or gPSCs to various tissues in chimeras was confirmed by standard DNA microsatellite genotyping analysis using D12Mit136 primers ( Table S2 ). Chimeras were mated with albino strain ICR mice to further examine their germline transmission competence.

Follicle Count Liu et al., 2013 Liu M.

Yin Y.

Ye X.

Zeng M.

Zhao Q.

Keefe D.L.

Liu L. Resveratrol protects against age-associated infertility in mice. The aggregate-formed rOvaries were carefully retrieved and subsequently dehydrated with graded alcohols, cleared in xylene, and embedded in paraffin wax. The serial sections (5 μm) from each rOvary were aligned in order on glass microscope slides, stained with H&E and analyzed for the number of follicles at different developmental stages in every fifth section with random start in the first five sections. The total number of follicles per rOvary was calculated by combining the counts of every fifth section throughout the whole rOvary (). The follicles were categorized into primordial or primary, secondary, and antral or mature and atretic accordingly. Primordial, primary, and intermediate-stage follicles were identified by the presence of an oocyte surrounded by a single layer of flat, squamous, or cuboidal cells. Secondary follicles were characterized as having more than one layer of GCs with no visible antrum. Antral or mature follicles possessed small areas of follicular fluid (antrum) or a single large antral space. Only those follicles containing an oocyte with a clearly visible nucleus were scored.

Immunofluorescence Microscopy Cells were washed twice in PBS, fixed in freshly prepared 3.7% paraformaldehyde for 30 min on ice, washed once in PBS and permeabilized in 0.1% Triton X-100 in blocking solution (3% goat serum plus 0.1% BSA in PBS) for 30 min at room temperature, then washed three times in PBS, and left in blocking solution for 2 h. Cells were incubated overnight at 4°C with primary antibodies anti-Oct4, Nanog, Gata4, LaminA, Foxl2, Ki67, H3K27me3, βIII-tubulin, AFP, α-SMA, Nestin, Zscan4, or Pan-Kcr in blocking solution. Cells were washed three times (each for 15 min) with blocking solution, and incubated for 2 h with secondary antibodies at room temperature. FITC-goat anti-mouse IgG (H+L), Alexa Fluor® 594 goat anti-rabbit IgG (H+L), or Alexa Fluor® 594 donkey anti-goat IgG (H+L) diluted 1:200 with blocking solution were used. Samples were washed, and counterstained with 0.5 μg/ml DAPI (Roche) in Vectashield (Vector) mounting medium. Fluorescence was detected and imaged using Axio-Imager Z2 Fluorescence Microscope (Carl Zeiss).

Fluorescence Microscopy of Teratoma or Tissue Sections Briefly, after being deparaffinized, rehydration, and wash in 0.01 M PBS (pH = 7.2), sections were subjected to high pressure antigen recovery sequentially in 0.01 M citrate buffer (pH = 6.0) for 3 min, incubated with blocking solution (5% goat serum and 0.1% BSA in PBS) for 2 h at room temperature, and then with the diluted primary antibodies overnight at 4°C. The following primary antibodies were used for immunocytochemistry: anti-Nestin, α-SMA and AFP. Blocking solution without the primary antibody served as negative control. After washing with PBS, sections were incubated with appropriate secondary antibodies, FITC-goat anti-mouse IgG (H+L) or AlexaFluor® 594 goat anti-rabbit IgG (H+L). Nuclei were stained with 0.5 μg/ml DAPI in Vectashield mounting medium. Fluorescence was detected and imaged using Axio-Imager Z2 Fluorescence Microscope (Carl Zeiss).

Fluorescence Microscopy of Meiocyte Spreads Liu et al., 2004 Liu L.

Franco S.

Spyropoulos B.

Moens P.B.

Blasco M.A.

Keefe D.L. Irregular telomeres impair meiotic synapsis and recombination in mice. Liu et al., 2004 Liu L.

Franco S.

Spyropoulos B.

Moens P.B.

Blasco M.A.

Keefe D.L. Irregular telomeres impair meiotic synapsis and recombination in mice. Surface spreading of meiocytes was prepared by a drying-down technique and stained for synaptonemal complexes (). PGC-aggregates, PGCLC-aggregates and E17.5 ovaries were collected, minced with two forceps and dissociated by pipetting in 0.05% TE. After incubation for 7 min at 37°C, cell suspensions were mixed with an equal volume of FBS, centrifuged for 5 min and resuspended in 100 mM sucrose. The cell suspension was spread onto glass slide by dipping onto a thin layer of fixative (1% paraformaldehyde, 0.15% Triton X-100 and 3 mM dithiothreitol, pH = 9.2). The glass slides were maintained overnight in a humidified box at 4°C. The slides were washed in water containing 0.4% Photo-flow (Kodak), and completely dried at room temperature. Dried slides were washed with 0.1% Triton X-100/PBS (PBST) for 10min, and incubated with antibody dilution buffer (ADB, 3% BSA, 2% goat serum/PBST) for 1 h at room temperature. Spreads were then incubated with anti-SCP1, SCP3 or MLH1 antibody in ADB at 4°C overnight, washed three times, then incubated with appropriate secondary antibodies, AlexaFluor® 488 Goat anti-Rabbit IgG (H+L), AlexaFluor® 594 Goat anti-Rabbit IgG (H+L), FITC Goat anti-Mouse IgG (H+L) or AlexaFluor® 594 Goat anti-Mouse IgG (H+L), washed, added with DAPI, and mounted in Vectashield mounting medium (Vector Laboratories). Immunofluorescence was detected using Axio-Imager Z2 Fluorescence Microscope. MLH1 foci were counted as described ().

Immunofluorescence Microscopy of Spindle Allworth and Albertini, 1993 Allworth A.E.

Albertini D.F. Meiotic maturation in cultured bovine oocytes is accompanied by remodeling of the cumulus cell cytoskeleton. 3 , 0.01% Triton X-100, 0.2% non-fat dry milk, 2% goat serum, 2% BSA and 0.1 M glycine). Afterward, oocytes were incubated with anti-α-Tubulin-FITC antibody at 37°C for 2 h. After washing, samples were stained for actin filaments with Texas Red-conjugated Phalloidin (1:1000) for 30 min, washed again and counterstained with 0.5 μg/ml DAPI (Roche) in Vectashield (Vector) mounting medium. Fluorescence was detected and imaged using Axio-Imager Z2 Fluorescence Microscope (Carl Zeiss). Tubulin, actin filament and chromatin were stained and observed by immunofluorescence microscopy, as described previously (). Denuded MII oocytes were fixed and extracted for 30 min at 37°C in a microtubule-stabilizing buffer. Oocytes were washed extensively and blocked overnight at 4°C in wash medium (PBS, supplemented with 0.02% NaN, 0.01% Triton X-100, 0.2% non-fat dry milk, 2% goat serum, 2% BSA and 0.1 M glycine). Afterward, oocytes were incubated with anti-α-Tubulin-FITC antibody at 37°C for 2 h. After washing, samples were stained for actin filaments with Texas Red-conjugated Phalloidin (1:1000) for 30 min, washed again and counterstained with 0.5 μg/ml DAPI (Roche) in Vectashield (Vector) mounting medium. Fluorescence was detected and imaged using Axio-Imager Z2 Fluorescence Microscope (Carl Zeiss).

Hormone Assays Serum follicle-stimulating hormone (FSH), estradiol (E2) and anti- Müllerian hormone (AMH) levels were assayed by ELISA kit (CK-E20381, CK-E20419 and CK-E90200, Hangzhou EastBiopharm CO., LTD). Quality control serum, sterilized distilled water, and five series diluted standard samples for a standard curve were tested for each serum sample. The intra- and inter-assay coefficients of variability for AMH, FSH, and E2 were below 8% and 12%.

Gene Expression Analysis by Real-Time qPCR Total RNA was purified using RNA mini kit (QIAGEN), treated with DNase I (QIAGEN), and the cDNA was generated from 2 μg RNA using Oligo (dT)18 primer (Takara) and M-MLV Reverse Transcriptase (Invitrogen). Real-time quantitative PCR (qPCR) reactions were set up in duplicate with the FS Universal SYBR Green Master (Roche) and carried out on an iCycler MyiQ2 Detection System (Bio-Rad). All reactions were carried out by amplifying target genes and internal control in the same plate. Each sample was repeated three times and normalized using Gapdh as the internal control. The amplification was performed for primary denaturation at 95°C for 10 min, then 40 cycles of denaturation at 95°C for 15 s, annealing and elongation at 58°C for 1 min, and the last cycle under 55-95°C for dissociation curve. Relative quantitative evaluation of target gene was determined by comparing the threshold cycles. Primers ( Table S3 ) were confirmed for their specificity with dissociation curves.

Western Blot Cells were washed twice in PBS, collected, and lysed in cell lysis buffer on ice for 30 min and then sonicated for 1 min at 60 of amplitude at 2 s intervals. After centrifugation at 10,000 g at 4°C for 10 min, supernatant was transferred into new tubes. The concentration of the protein sample was measured by bicinchoninic acid, and protein samples boiled in SDS sample buffer at 95°C for 10 min. Protein (10 μg) of each cell extract was resolved by 10% Acr-Bis SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Millipore). Nonspecific binding was blocked by incubation in 5% skim milk or 5% BSA in TBST at room temperature for 2 h. Blots were then probed with primary antibodies overnight by incubation at 4°C with Oct4, Nanog, Sox2, Lin28, Zscan4, Pan-Kcr, LaminA, PCNA, Bcl-2, Bcl-xL, Bad, Bax or β-Actin served as loading control. Immunoreactivity bands were then probed for 2 h at room temperature with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies, HRP-goat anti-rabbit IgG, or HPR-goat anti-mouse IgG (H+L). Protein bands were detected by Chemiluminescent HRP substrate (WBKLS0500, Millipore).

DNA Methylation in Promoter Region by Bisulfite Sequencing Genomic DNA was extracted from samples of GCs, ESCs and gPSCs using DNeasy Tissue Kit (QIAGEN) according to the manufacturer’s instructions. Bisulfite treatment of DNA was performed with the EpiTect Bisulfite Kit (QIAGEN). Bisulfite converted DNA was amplified by PCR, using HS Taq DNA Polymerase (QIAGEN). Thermal cycling was carried out with a 10 min denaturation step at 94°C, followed by 35 three-step cycles (30 s at 94°C, 30 s at 55-58°C and 30 s at 72°C) and final incubation at 72°C for 10 min. PCR products were recovered from stained gels (QIAquick Gel Extraction Kit, QIAGEN), cloned into a pGEM-T Easy vector (Promega) and then sequenced using T7 or SP6 primers using an ABI 3730 capillary genetic analyzer (Applied Biosystems) by BigDye terminator sequencing chemistry. Bisulfite efficiency as the fraction of modified cytosines in non-CpG sequences exceeded 98%.

Xist RNA-FISH Xist RNA-FISH was performed according to the manufacturer’s instructions (Biosearch Technologies). Growing cells were washed with PBS and fixed with 10% formaldehyde for 10 min at room temperature. After being washed twice with PBS, cells were immersed in 70% ethanol for at least one hour at 4°C. Wash Buffer A (SMF-WA1-60, Biosearch Technologies) were added to cells and incubated at room temperature for 5 min. After incubation with the Hybridization Buffer (SMF-HB1-10, Biosearch Technologies) containing Xist RNA-FISH probe (SMF-3011-1, Biosearch Technologies) for 12-16 h at 37°C, cells were washed with Wash Buffer A and Wash Buffer B (SMF-WA1-20, Biosearch Technologies). Nuclei were stained with 0.5 μg/ml DAPI in Vectashield mounting medium. Fluorescence was detected and imaged using Axio-Imager Z2 Fluorescence Microscope.

Telomerase Activity by TRAP Assay Telomerase activity was determined by the Stretch PCR method according to manufacturer’s instruction using TeloChaser Telomerase assay kit (T0001, MD Biotechnology). About 2.5 × 104 cells from each sample were lysed. Lysis buffer served as negative controls. PCR products of cell lysate were separated on non-denaturing TBE-based 12% polyacrylamide gel electrophoresis and visualized by ethidium bromide (EB) staining.

Telomerase Assay by ELISA Telomerase level was determined by ELISA method according to manufacturer’s instruction using Mouse Telomerase (TE) ELISA kit (CSB-E08022m, CUSABIO).

Telomere Measurement by Real-Time qPCR Cawthon, 2002 Cawthon R.M. Telomere measurement by quantitative PCR. Liu et al., 2007 Liu L.

Bailey S.M.

Okuka M.

Muñoz P.

Li C.

Zhou L.

Wu C.

Czerwiec E.

Sandler L.

Seyfang A.

et al. Telomere lengthening early in development. Genome DNA was prepared using DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA). Average telomere length was measured from total genomic DNA using a real-time PCR assay (). PCR reactions were performed on the iCycler iQ5 2.0 Standard Edition Optical System (Bio-Rad, Hercules, CA), using telomeric primers, primers for the reference control gene (mouse 36B4 single copy gene) and PCR settings as previously described () ( Table S4 ). For each PCR reaction, a standard curve was made by serial dilutions of known amounts of DNA. The telomere signal was normalized to the signal from the single copy gene to generate a T/S ratio indicative of relative telomere length. Equal amount of DNA (20 ng) was used for each reaction.

Telomere Restriction Fragment Measurement The telomere restriction fragment (TRF) analysis was performed using a commercial kit (TeloTAGGG Telomere Length Assay, cat no. 12209136001, Roche). DNA was extracted from cells by phenol-chloroform method. A total of 3 μg DNA was digested overnight with MboI endonuclease (NEB) at 37°C and electrophoresed through 1% agarose gels in 0.5 × TBE at 14°C for 16 h at 6 V/cm with an initial pulse time of 1 s and end in 12 s using a CHEF Mapper pulsed field electrophoresis system (Bio-Rad). The gel was blotted and probed using reagents in the kit. The telomere length is quantified by TeloTool software (Available from https://www.mathworks.com/matlabcentral/fileexchange/44573-telotool-terminal-restriction-fragment-analysis ).

Telomere Q-FISH Herrera et al., 1999 Herrera E.

Samper E.

Martín-Caballero J.

Flores J.M.

Lee H.W.

Blasco M.A. Disease states associated with telomerase deficiency appear earlier in mice with short telomeres. Poon et al., 1999 Poon S.S.

Martens U.M.

Ward R.K.

Lansdorp P.M. Telomere length measurements using digital fluorescence microscopy. 3 peptide nucleic acid (PNA) probe at 0.5 μg/ml (F1001, Panagene, Korea). Chromosomes were stained with 0.5 μg/ml DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss microscope with FITC/DAPI or Cy3/DAPI using AxioCam and AxioVision software 4.6. Telomere length shown as telomere fluorescence intensity was integrated using the TFL-TELO program (kindly provided by Peter Lansdorp, Terry Fox Laboratory). Telomere length was estimated by telomere Q-FISH as described previously (). Telomeres were denatured at 80°C for 3 min and hybridized with FITC- or Cy3-labeled (CCCTAA)peptide nucleic acid (PNA) probe at 0.5 μg/ml (F1001, Panagene, Korea). Chromosomes were stained with 0.5 μg/ml DAPI. Fluorescence from chromosomes and telomeres was digitally imaged on a Zeiss microscope with FITC/DAPI or Cy3/DAPI using AxioCam and AxioVision software 4.6. Telomere length shown as telomere fluorescence intensity was integrated using the TFL-TELO program (kindly provided by Peter Lansdorp, Terry Fox Laboratory).

Flow-FISH Analysis of Telomeres Baerlocher and Lansdorp, 2003 Baerlocher G.M.

Lansdorp P.M. Telomere length measurements in leukocyte subsets by automated multicolor flow-FISH. Canela et al., 2007 Canela A.

Vera E.

Klatt P.

Blasco M.A. High-throughput telomere length quantification by FISH and its application to human population studies. 3 peptide nucleic acid (PNA) probe at 0.5 μg/ml (F1002, Panagene, Korea). Then cells were shaken and washed three times and FACS analysis performed using a Flow Cytometer (BD Biosciences). Flow-FISH analysis of telomeres was performed as described previously (). Cells suspension were fixed with 70% alcohol for 10 min at 4°C, and dehydration in 85% alcohol and 100% alcohol. Telomeres were denatured at 80°C for 3 min and hybridized with Cy3-labeled (CCCTAA)peptide nucleic acid (PNA) probe at 0.5 μg/ml (F1002, Panagene, Korea). Then cells were shaken and washed three times and FACS analysis performed using a Flow Cytometer (BD Biosciences).

Library Preparation and RNA-Sequencing mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5x). First strand cDNA was synthesized using random hexamer primer and M-MLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3′ ends of DNA fragments, NEB Next Adaptors with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150∼200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μL USER Enzyme (NEB, USA) was used with size-selected and cDNA adaptor ligated at 37°C for 15 min followed by 5 min at 95°C prior to PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer. At last, PCR products were purified using AMPure XP system and library quality assessed on the Agilent Bioanalyzer 2100 system. Cluster of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform. Picelli et al., 2014 Picelli S.

Faridani O.R.

Björklund A.K.

Winberg G.

Sagasser S.

Sandberg R. Full-length RNA-seq from single cells using Smart-seq2. For Library preparation of a few cells, 1000 cells per sample were resuspended in PBS with 0.1% BSA and transferred to the bottom of a PCR tube consisting of 3 μL lysis buffer, and cDNA was synthesized in the tube containing mRNA, based on Smart-seq2 protocol (), and then the libraries were prepared by using TruePrep DNA Library Prep Kit V2 for Illumina® (TD503-02, Vazyme Biotech) according to the manual instruction. Samples were barcoded and multiplex sequenced with a 150-bp paired-end sequencing strategy on an Illumina Hiseq platform.

Bioinformatics Analysis Hochberg and Benjamini, 1990 Hochberg Y.

Benjamini Y. More powerful procedures for multiple significance testing. 2 (fold change) of 1 were set as the threshold for significantly differential gene expression. Cluster and analysis of XEN-like, 2C, pluripotency, PGCs specific and germ cell genes provided in the heatmap were obtained using RNA-seq data published ( Hayashi et al., 2012 Hayashi K.

Ogushi S.

Kurimoto K.

Shimamoto S.

Ohta H.

Saitou M. Offspring from oocytes derived from in vitro primordial germ cell-like cells in mice. Miyauchi et al., 2017 Miyauchi H.

Ohta H.

Nagaoka S.

Nakaki F.

Sasaki K.

Hayashi K.

Yabuta Y.

Nakamura T.

Yamamoto T.

Saitou M. Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice. Zhao et al., 2015 Zhao Y.

Zhao T.

Guan J.

Zhang X.

Fu Y.

Ye J.

Zhu J.

Meng G.

Ge J.

Yang S.

et al. A XEN-like State Bridges Somatic Cells to Pluripotency during Chemical Reprogramming. Zhao et al., 2018 Zhao T.

Fu Y.

Zhu J.

Liu Y.

Zhang Q.

Yi Z.

Chen S.

Jiao Z.

Xu X.

Xu J.

et al. Single-Cell RNA-Seq Reveals Dynamic Early Embryonic-like Programs during Chemical Reprogramming. Clean reads were mapped to the mouse reference mm10 reference genome using Bowtie2 (population) or Hisat2 (a few cells). Reads were assigned and counted to genes using the RSEM. The resulting matrix of read counts was then loaded into RStudio (R version 3.4.2), and DESeq2 were used to identify differentially expressed genes. Functional enrichment (GO annotation or KEGG) of gene sets with different expression patterns was performed using clusterProfiler. The heat-maps were drawn by the function “pheatmap” of R packages and correlation coefficients were calculated by the function “cor” in R. Scatterplots were generated using the “ggplot2” package to graphically reveal genes that differ significantly between two samples. The p values were adjusted using the Benjamin & Hochberg method (). Corrected P-value of 0.05 and log(fold change) of 1 were set as the threshold for significantly differential gene expression. Cluster and analysis of XEN-like, 2C, pluripotency, PGCs specific and germ cell genes provided in the heatmap were obtained using RNA-seq data published (). Calculated z score of selected genes was used for heatmap.

Whole-Exome Sequencing For population cells, paired-end DNA library were prepared according to manufacturer’s instructions (Agilent). Genomic DNAs (gDNA) from cell samples were sheared into 180∼280 bp fragments by Covaris S220 sonicator. Ends of gDNA fragments were repaired and 3′ ends were adenylated. Both ends of gDNA fragments were ligated at the 3′ ends with paired-end adaptors (Illumina) with a single ‘T’ base overhang and purified using AMPure SPRI beads from Agencourt. The adaptor-modified gDNA fragments were enriched by six cycles of PCR using SureSelect Primer and SureSelect ILM Indexing PreCapture PCR Reverse Primer. The concentration and size distribution of the libraries were determined on an Agilent Bioanalyzer DNA 1000 chip. Whole exome capture was carried out using SureSelect Mouse All Exon V1 Agilent 5190-4642. An amount of 0.5 μg prepared gDNA library was hybridized with capture library for 5 min at 95°C followed by 24 h at 65°C. The captured DNA−RNA hybrids were recovered using Dynabeads MyOne Streptavidin T1. Capture products were eluted from the beads and desalted using QIAGEN MinElute PCR purification columns. The purified capture products were then amplified using the SureSelect ILM Indexing Post Capture Forward PCR Primer and PCR Primer Index (Agilent) for 12 cycles. After DNA quality assessment, captured DNA library were sequenced on Illumina Hiseq 2000 sequencing platform (Illumina) according to manufacturer’s instructions for paired-end 150 bp reads (Novogene). Libraries were loaded onto paired-end flow cells at concentrations of 14-15 pM to generate cluster densities of 800,000-900,000 per mm2 using Illumina cBot and HiSeq paired-end cluster kit. Zong et al., 2012 Zong C.

Lu S.

Chapman A.R.

Xie X.S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. For GV oocytes, 10 GV oocytes per sample were pre-amplified and amplification based on the method of multiple annealing and looping based amplification cycles (MALBAC) described previously (). Briefly, 30 μL of amplification buffer was added into PCR tubes each containing 10 lysed GV oocytes, followed by incubating the tube at 94°C for 3 min to melt the double-stranded genome DNA into single-stranded form. After melting, the single-stranded genomic DNA molecules were immediately quenched on ice to increase the efficiency of primer binding. Then, 2.5 Units of Bst large fragment (NEB) were added into each PCR tube and the following temperature steps were performed: 10°C for 45 s, 20°C for 45 s, 30°C for 45 s, 40°C for 45 s, 50°C for 45 s, 65°C for 2 min, 94°C for 20 s. The tubes were then quickly quenched on ice. The same polymerase mix was added to provide enzyme for the next round of amplification. The following thermo-cycle was performed before quenching the reactions on ice: 10°C for 45 s, 20°C for 45 s, 30°C for 45 s, 40°C for 45 s, 50°C for 45 s, 65°C for 2 min, 94°C for 20 s, 58°C for 20 s (cycles were repeated 5 times). The product from the MALBAC pre-amplification was further amplified by regular PCR using the following thermal cycle repeated 18 times: 94°C for 20 s, 59°C for 20 s, 65°C for 1 min, and 72°C for 2 min. The amplification products had a size distribution of about 500 bp to 1500 bp, and these products can be used to build DNA library and sequencing using the method mentioned above.