All procedures were implemented according to the Local Experimental Animal Care Committee and approved by the ethics committee of Nanjing Agricultural University (Nanjing, China; SYXK-2019-00085).

Fertilized Hyline chicken eggs were incubated at 38.5°C and 60% humidity in an egg incubator (BSS160; Grumbach, Mücke, Germany) according to the standard operating protocols. PGCs were isolated from embryonic genital ridges at E4.5 using our standard protocol [23]. The culture medium was α-MEM (1257107; Gibco, Thermo Fisher Scientifc, Waltham, MA, USA) supplemented with 15% knockout serum replacement (KSR, 10828-028; Invitrogen, Carlsbad, CA, USA), 10 ng/mL leukemia inhibitory factor, 10 ng/mL human bFGF (060-04543; Wako Pure Chemicals, Osaka, Japan), and 2% chicken embryo extract. Chicken PGCs were cultured in an incubator maintained at 37°C with an atmosphere of 5% CO2 and 60% to 70% relative humidity. Anti-stage-specific embryonic antigen (SSEA-1) antibody was used to identify and sort PGCs.

To test the role of Wnt4/β-catenin in Tet1-mediated PGC proliferation, specific activator 6-bromoindirubin-3′-oxime (BIO, 0, 0.5, 1.0, or 1.5 μM) and inhibitor JW74 (0, 5, 10, or 15 μM) (Sigma-Aldrich Chemical Company, St.Louis, MO, USA) were used to detect their effects. BIO and JW74 were dissolved in dimethyl sulfoxide (DMSO), and the control received the vehicle only with a final DMSO concentration of <0.1% that had no significant effect on cell proliferation and survival.

For RNA interference assays, Tet1-specific siRNAs were designed according to our previous study [5]. Cells were plated in 12-well plates at 5×10 cells per well and transfected with 50 nM indicated siRNAs plus siRNA mate (GenePharma, Shanghai, China) according to the manufacturer’s protocol. PGCs were sorted with fluorescence-activated cell sorting (FACS) after transfection and replated for 48 h before being harvested and used for characterizations and further measurement.

Total RNA was extracted from each sample with Trizol reagent (Invitrogen, USA). Quality and purity were determined by the Nanodrop-2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and the integrity was detected by 1.5% agarose gel-electrophoresis. RNA (500 ng) was reverse transcribed to cDNA after DNase treatment by using 5× All-In-One RT MasterMix (Applied Biological Materials Inc., Richmond, BC, Canada) according to the manufacturer’s instructions. Quantitative real time polymerase chain reaction (qRT-PCR) was performed by using SYBR Premix Ex TaqTM kit (Takara, Dalian, China) on an ABI7500 Fast thermal cycler (Applied Bio-systems Biosystems, Foster City, CA, USA) by following the manufacturer’s instructions. The final PCR reactions contained 10 μL 2×SYBR Premix Ex Taq II (Tli RNaseH Plus), 0.8 μL of forwards and reverse primer (10 mM), 2 μL first-strand cDNA and 6.4 μL H₂O. Cycling parameters were 95°C for 30 s, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. Primer specificity was determined by melt curve analysis. Both no-reverse-transcription and no-template controls were used to confirm a lack of genomic DNA contamination and primer dimerization. All samples were normalized with the β-actin using the comparative cycle threshold method (2^–ΔΔCt). Primer sequences are listed in Table 1. Differences with p<0.05 were considered as significantly different.

Chicken PGCs were grown on gelatinised glass coverslips, washed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde for 20 min at room temperature (RT). Cells were permeabilized with 0.55 Triton X-100 in PBS for 30 min at RT, washed with PBS and saturated with 2% bovine serum albumin (BSA) in PBS for 1 h. Then PGCs were incubated with the primary antibody raised against anti-SSEA-1 antibody (1:1,000; Developmental Studies Hybridoma Bank, Iowa, IA, USA), 5mC and 5hmC (1:1,000; Abcam, Cambridge, UK) overnight at 4°C in the blocking solution. After three washes with 0.1% Tween-20 in PBS, PGCs were incubated in Alexa-IgG-568 antibodies, fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin M (IgM), or goat anti-mouse IgG (1:1,000; KPL Inc., Gaithersburg, MD, USA) as the secondary antibody in the blocking solution for 1 h. The nuclei were counterstained with 4′, 6-diamidino-2-phenylindole (DAPI, 300 nm; Sigma Aldrich, St. Louis, MO, USA). After a brief wash in PBS, samples were mounted with SlowFade Gold antifade reagent (Invitrogen, USA). For the detection of 5mC and 5hmC, nuclear DNA was denatured with 2 N HCl for 30 min and then neutralized with 100 mM Tris-HCl (pH 8) for 10 min. Finally, the images were visualized using laser-scanning confocal microscopy (Zeiss LSM 700; Carl Zeiss AG, Oberkochen, Germany).

PGCs were sorted by FACS after treatment and lysed in radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitors (Complete; Roche, Shanghai, China) and 0.2 nM phenylmethyl sulfonyl fluoride. Protein (20 ng) was denatured and subjected to 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis. After transfer to polyvinylidene fluoride membrane, the membrane was incubated in 5% dry skim milk in tris buffered saline (0.1% Tween-20) at RT for 1 h. The primary antibodies were rabbit anti-phospho β-catenin (1:1,000; Abcam, UK), rabbit anti-WNT4 (1:1,000; Abcam, UK) and β-actin (1:5,000; Novus Biologicals, Littleton, CO, USA) at 4°C overnight. After 1 h incubation with anti-mouse IgG or anti-rabbit IgG peroxidase-conjugated second antibody (1:5,000; Santa Cruz, Santa Cruz, CA, USA), blots were developed by enhanced chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) and exposed to a ChemiDoc-XRS System (Bio-Rad, Hercules, CA, USA) to detect chemiluminescence.

The proliferation of PGCs was determined using the cell counting kit-8 (CCK8) assay (Dojindo, Kyushu, Japan) according to the manufacturer’s protocol. Briefly, 1×10⁴ cells were seeded into 96-well plate and transfected with siRNAs. After transfection for 48 h, fresh culture medium with 10 mL CCK8 solution was added and incubated for another 2 h at 37°C. The optical density (OD) of each well was measured at 450 nm using an automated microplate reader (Thermo Scientific Multiskan GO microplate spectrophotomete).

For the cell cycle analysis, PGCs were first dissociated into single cells and fixed in 70% ethanol at −20°C for 30 min after washing with ice-cold PBS. The cells were incubated with staining solution (50 μg/mL propidium iodide, 0.1% Triton X-100 and 100 mg/mL RNase A in PBS) for 15 min at 4°C. The stained cells were analyzed by flow cytometry (BD FACSCELLULAR, Franklin Lakes, NJ, USA). Data was analyzed using FACSDiva and Modfit LT cell-cycle analysis software (Verity Software House, Topsham, ME, USA).

To detect 5mC and 5hmC levels, genomic DNA was extracted by phenol-chloroform-isoamyl alcohol, and the concentration of samples was serially diluted twofold. Dot blot analysis was performed as described previously [5]. Subsequently, genomic DNA was spotted on a positively charged nylon membrane (Roth, Karlsruhe, Germany), air dried for 15 min and cross-linked using the UV light (20 s, 1,200 J/cm²). Then membranes were blocked in 5% non-fat milk in PBS/0.1% Tween-20 for 1 h at RT before incubation incubated with anti-5mC (1:5,000; Abcam, UK) antibody or anti-5hmC (1:5,000; Active Motif, Carlsbad, CA, USA) overnight at 4°C followed by washing in PBS for 3 times. The membrane was then incubated with HRP-labeled anti-mouse/rabbit secondary antibody (1:5,000; Santa Cruz, USA) and signal was developed using an enhanced chemiluminescence reagent (Bio-Rad, USA). The intensity of signal was measured using Image J software and calibrated against the linear range of the standard curves to estimate the quantities of 5mC or 5hmC in each sample.

Statistical analysis was performed using SAS version 9.4 software (SAS Institute, Cary, NC, USA). When a significant main effect was detected by analysis of variance using SAS, the least-squares analysis was used to compare the different treatments. Differences between control and treatment groups were deemed to be significant when p<0.05.

To investigate the role of Tet1 in the pluripotency of PGCs, siRNA targeting Tet1 was used to transiently knockdown of Tet1 in chicken PGCs. Both mRNA and protein expression of Tet1 in PGCs was examined after Tet1 siRNA transfection for 48 h. The results demonstrated that the mRNA expression level of Tet1 decreased approximately by 50% in Tet1 knockdown group compared with control (Figure 1A) (p< 0.01). In addition, the reduction of TET1 protein was observed by western blotting analysis (Figure 1B). Introduction of siRNA resulted in a 3-fold reduction in the level of TET1 (Figure 1C) (p<0.01). Furthermore, the expression of Nanog, PouV, and Sox2 was declined significantly after the down-regulation of Tet1 (Figure 1D) (p<0.05).

To address the involvement of Tet1 in the level of genomic DNA methylation, the status of 5mC and 5hmC in PGCs was measured by immunostaining and DNA dot blot. Results showed that knockdown of Tet1 led to a general increase in level of 5mC and the level of 5hmC diminished in PGCs (Figure 2A). A similar distribution of 5mC and 5hmC in PGCs was confirmed by DNA dot blot analysis. Expectedly, it was found that Tet1 knockdown induced significant increase in 5mC level in siTet1 group compared with control (Figure 2B–2C) (p<0.01). By contrast, knockdown of Tet1 induced significant decrease 5hmC level in siTet1 group (Figure 2B–2C) (p<0.01). This suggested that the potent capacity of PGCs to induce demethylation was restricted in Tet1 knockdown group.

In this study, the level of β-catenin was detected to investigate the possible mechanism underlying the activity of Tet1. The culture medium was added with 0.5, 1.0, and 1.5 μM BIO or 5.0, 10, and 15 μM JW74. Result revealed that only a modest increase in the level of β-catenin was observed in 0.5 μM BIO, while 1 μM BIO showed substantial increase in β-catenin level (Figure 3A) (p<0.05). By contrast, the level of β-catenin remarkably reduced at 10 μM and 15 μm JW74 treated PGCs (Figure 3B) (p<0.05). The level of β-catenin was rescued in Tet1 knockdown group with addition of BIO, but it was still downregulated significantly compared with control (Figure 3C) (p<0.05).

To determine whether Tet1 exerted the proliferative effect through Wnt4/β-catenin signaling pathway, siTet1 was added along with BIO in PGC culture and the cell-cycle phase distribution was first measured. Results indicated that a notable decrease in G2/M phase was observed in cells treated with siTet1 and JW74 (Figure 4A) (p<0.05), demonstrating that PGC proliferation was significantly inhibited by JW74. By contrast, cell cycle distribution analysis showed the accumulation of cells in S and G2/M phase and a decrease in the proportion of cells in G1 phase after incubation with BIO (Figure 4A) (p<0.05). Cell counting showed there to be less cell proliferation in BIO and siTet1 than that in control group or group treated with BIO alone (Figure 4A) (p<0.05), indicating the proliferative effect of Tet1 on chicken PGCs was induced via the Wnt4/β-catenin signaling pathway. Moreover, the proliferative capacity of cultured PGCs was examined by using the CCK8 assay. As shown in Figure 4B, the growth rate of the siRNA and JW74 group decreased significantly compared with the control group, while the BIO and siTet1 combined with BIO group increased PGC proliferation significantly (p<0.05). To further elucidate the effect of Tet1 knockdown on PGCs, the expression of cell cycle and pluripotency related genes was examined. Tet1 knockdown blocked the mRNA expression of cyclin D1/CDK6, and consequently delayed the entrance to G1/S phase (p<0.05) (Figure 4C). However, this inhibition was rescued by the addition of BIO. To further investigate the role of Tet1 in the pluripotency of PGCs via Wnt4/β-catenin signaling pathway, the expression of pluripotency related genes Nanog, Sox2, and PouV was evaluated, in siTet1 and JW74 group, all those three genes were significantly downregulated, while they were significantly elevated in BIO and siTet1 combined with BIO group (Figure 4D) (p<0.05). These results, along with the dot blot results shown previously, confirmed that Tet1 induced the accumulation of 5hmC in PGCs to promote their proliferation and knockdown of Tet1 in PGCs led to inhibiting cell growth via Wnt4/signaling pathway.

To investigate whether Tet1 is required for maintaining the hypomethylated state, the abundance of 5mC and 5hmC in Tet1 knockdown PGCs was estimated by using dot blot. Result reflected an obviously trend for increasing 5mC level in Tet1 knockdown PGCs was observed (Figure 5A) (p<0.05). Tet1 knockdown as well as JW74 significantly impaired the accumulation of 5hmC, whereas addition of BIO resulted in a marked increase in 5hmC level (Figure 5B) (p<0.05). After disrupting the level of 5hmC in PGCs by down-regulating Tet1, there was no significant change of 5hmC status in BIO and siTet1 combined BIO group (Figure 5B) (p<0.05). As shown in Figure 5B, treatment with siTet1 as well as JW74 prevented 5hmC accumulation in PGCs (p<0.05).

Next, whether histone demethylation plays any roles in chicken PGCs, the protein level of H3K9me2 and H3K27me3 were probed by western blot. Interestingly, the H3K27me3 global level was elevated, whereas the H3K9me2 level was greatly reduced in siTet1 group compared to control group, but BIO reduced H3K27me3 level and elevated H3K9me2 level (Figure 5C) (p<0.05). In addition, siTet1 with BIO elevated both H3K9Mme2 and H3K27me3 (Figure 5C) (p< 0.05). Moreover, we found that the level of H3K27me3 was important for chicken PGC proliferation.