Several sexually mature (30 weeks old) Jining Bairi hens, an indigenous chicken breed that is well known for early sexual maturity at around 100 days, were randomly selected from the local research farm affiliated with Shandong Agricultural University. All chickens had free access to water and feed. The chickens were housed in separate cages with a daily light period of 14 h and the laying events were monitored to determine the regular laying sequence. Three hens in the middle of a laying sequence were sacrificed by decapitation immediately after oviposition. All of the small yellow follicles (6–8 mm in diameter) were collected and stored in liquid nitrogen for RNA isolation. All of the animal experiments were approved by the Institutional Animal Care and Use Ethics Committee of Shandong Agricultural University and performed in accordance with the “Guidelines for Experimental Animals” of the Ministry of Science and Technology of China.

Total RNA was extracted from all of the small yellow follicles (6–8 mm) using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA purification, cDNA synthesis, and RNA-seq were performed at Biomarker Technologies Co., Ltd. (Beijing, China). Enrichment of eukaryotic mRNA was performed using a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490), and the rRNA and concentrated prokaryotic mRNA were removed using a MICROBExpress Bacterial mRNA Enrichment Kit (Invitrogen, AM1905). mRNA was used as a template to construct the library. The qualified library was clustered on an Illumina cBot and finally sequenced using an Illumina HiSeq 2500. Prior to analysis, the low-quality reads and rRNA sequences were removed, and the clean reads were mapped to the chicken genome. The structure and expression of DEGs were analyzed and the functional annotation of the genes was accomplished by comparison with the reference genome: Gallus gallus (assembly Gallus_gallus-5.0).

Protein–protein interaction networks were constructed based on the information from STRING version 10.0, which permits the critical assessment and integration of PPIs, including both direct (physical) and indirect (functional) associations. Credible interactions (combined_score ≥0.4) were accepted for further network analysis using CytoScape.

The hierarchical follicles and all of the small yellow follicles were isolated from egg-laying hens and placed in phosphate-buffered saline (PBS). The small yellow follicles were treated with 0.1% collagenase II (MP Biomedicals, Santa Ana, CA, USA) at 37°C for 10 min to obtain the GCs. The yolks of the follicles were removed carefully with ophthalmic forceps and the GCs were isolated from the hierarchical follicles and then dispersed by treatment with 0.25% trypsin–EDTA (Gibco) at 37°C for 15 min with gentle oscillation in a centrifuge tube. After centrifugation, the GCs were suspended in culture medium (M199 with 10% fetal bovine serum and 1% penicillin/streptomycin), and subsequently seeded in 24-well culture plates at a density of 1 × 10⁵/well. The number of cells was detected using Trypan blue. Cells were cultured at 38°C in an atmosphere of water-saturated 5% CO₂ for 24 h.

The coding DNA sequence region of Wnt4 was cloned from chicken cDNA by PCR. The amplified fragment spanned the region between the transcription initiation site and the termination codon. PCR products were cloned into the pUSEamp vector (Millipore) to generate the overexpression plasmid. All of the primers are listed in Table S1 in Supplementary Material. The shRNA of Wnt4 was synthesized by Shanghai GenePharma Co., Ltd. (Shanghai, China), and the target sequences are listed in Table S2 in Supplementary Material. GCs were plated on 24-well plates for the transient transfection experiments. The cells were transfected with the overexpression vector and shRNA (800 ng/well) using Lipofectamine LTX and Plus Reagent (Invitrogen). Six hours after transfection, recombinant FSH was added. Twenty-four hours after transfection, the cells were lysed for RNA and protein extraction.

The total RNA was extracted from all of the follicles using TRIzol reagent (Invitrogen), and the total RNA from the cultured GCs was isolated using a MicroElute Total RNA Kit (Omega). The quality of the total RNA samples was tested by gel electrophoresis and spectrophotometry. The cDNA was synthesized using a PrimeScript RT Reagent Kit with a gDNA Eraser (TaKaRa, Dalian, China). The total reaction volume was 20 µL, containing 1 µg total RNA, 1 µL oligo(dT)₁₈ primer, 1 µL of PrimeScript RT Enzyme Mix I, 4 µL of 5× PrimeScript Buffer, and RNase-free ddH₂O. The program was 37°C for 15 min and 85°C for 5 s. Real-time quantitative PCR was performed on an MX3000p system (Stratagene, La Jolla, CA, USA). The reaction system contained 1.5 µL cDNA, 7 µL of 2× SYBR Premix Ex Taq II (TaKaRa, Dalian, China), 0.3 µL of 50× Rox Reference Dye II, 0.3 µL of each of the forward and reverse primers, and RNase-free ddH₂O. The program was 95°C for 30 s, and 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 20 s. Melting curves were used to confirm the specificity of each product, and the PCR efficiency was determined by analysis of two-fold serial dilutions of the cDNA. The PCR efficiency was close to 100%, allowing the use of the 2^−ΔΔCT method for the calculation of relative gene expression (24). The mRNA expression of the genes FSHR, Wnt4, StAR, CYP11A1, AMH, OCLN, and so on was quantitatively analyzed by this method. All of the primers are listed in Table S1 in Supplementary Material.

The total protein content was isolated from different follicles using Cell Lysis Reagent (Fermentas) and the protein concentration was determined using the bicinchoninic acid assay. Goat anti-mouse Wnt4 polyclonal antibody was purchased from Santa Cruz Biotechnology, Inc. Equal amounts of protein were separated by 10% sodium dodecyl sulfate gel electrophoresis under denaturing and non-reducing conditions and then transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were first blocked, then incubated (1 h, 37°C) with the Wnt4 antibody diluted (1:500) in a 5% bovine serum albumin (BSA)/PBS solution. After washing in phosphate-buffered saline with Tween 20, the blots were incubated with horseradish-peroxidase-conjugated goat anti-mouse immunoglobulin G antibody (1:1,000; Abcam) in a 5% BSA/PBS solution. The signals were visualized using a 3,3′-diaminobenzidine (DAB) substrate kit (Tiangen).

The Wnt4 levels in the medium were detected using a Chicken Wingless-Type MMTV Integration Site Family Member 4 (Wnt4) ELISA Kit (Shanghai Enzyme-linked Biotechnology Co., Ltd.). The culture media of four kinds of cells [GCs and theca cells (TCs) of hierarchical follicles, and GCs and TCs of prehierarchical follicles] were collected and centrifuged at 3,000 rpm for 30 min to remove debris. The Wnt4 level was then determined according to the manufacturer’s instructions. The optical density of every well was tested at 450 nm using an ELx808 Absorbance Reader (BioTek, Winooski, VT, USA). The sample concentrations were calculated according to the mean absorbance.

The proliferation of GCs was detected using an Enhanced Cell Counting Kit-8 Assay Kit (Beyotime, Beijing, China). Approximately 6 × 10³ cells were seeded in every well of a 96-well plate. The cells were transfected with the pUSEamp-Wnt4 plasmid and shRNA of Wnt4 when the cells reached 60% confluence. At 0, 24, 48, and 72 h after transfection, 100 µL of medium with 10 µL of CCK8 was added to each well, and then the plates were incubated for a further 2 h at 38°C. The absorbance was evaluated using an ELx808 Absorbance Reader at 450 nm.

All data are presented as the mean ± SEM. The differences between different groups were determined by one-way ANOVA followed by Duncan’s test using the SPSS software (SPSS Inc., Chicago, IL, USA). The differences between groups were considered significantly different when p < 0.05.

RNA-seq was used to compare the transcriptomes of six small yellow follicles, which are referred to here as T1, T2, T3, T4, T5, and T6 (T1 and T4, T2 and T5, and T3 and T6 were derived from three different laying hens). The mRNA expression levels of FSHR for follicles T1, T4, T2, T5, T3, and T6 were 1.08 ± 0.01, 5.75 ± 0.27, 1.00 ± 0.06, 1.25 ± 0.16, 1.04 ± 0.19, and 1.08 ± 0.02, respectively, as determined by qRT-PCR, and the high-throughput RNA-seq generated 45.39, 44.14, 44.09, 43.81, 43.48, and 42.73 million raw reads, respectively, for the six follicles. Of these, 75.13–78.40% could be mapped to the chicken genome, and more than 98% of the reads were unique (Table 1). The sequencing reads were submitted to NCBI’s GEO under the accession number GSE100673.

A total of 17,993 genes were examined for the six follicles, and the expression of all of these genes is listed in Table S3 in Supplementary Material. In the process of screening for DEGs between groups, a fold change ≥2 and false discovery rate (FDR) <0.01 were used as the criteria. In the T1 vs. T4 group, 1,968 DEGs were detected, including 561 upregulated genes and 1,407 downregulated genes. In the T2 vs. T5 group, 289 DEGs were detected, including 104 upregulated genes and 185 downregulated genes. In the T3 vs. T6 group, 590 DEGs were detected, including 225 upregulated genes and 365 downregulated genes (Table S4 in Supplementary Material). To validate the RNA-seq data, some DEGs were chosen and quantified by qRT-PCR. The results showed that the mRNA levels of these genes were similar to the sequencing data (Table 2).

In order to explore the key candidate genes related to follicle development, a network of the DEGs was obtained using STRING PPI network analysis (Figure 1); all of the genes are listed in Table S5 in Supplementary Material. Some of the genes, for instance Smad2, Wnt4, and FSHR, were found to play vital roles in the network.

In the three groups, with the increase in FSHR expression, the number of DEGs increased. To further explore the biological functions of these genes, we performed KEGG analysis and GO assignments of the DEGs, and the corresponding information is listed in Tables S6 and S7 in Supplementary Material. The significantly enriched KEGG pathways of the three groups are shown in Table 3. In the T1 vs. T4 group, the Wnt signal pathway was significantly enriched, and the DEGs of the Wnt signal pathway are shown in Table 4. In the follicle with the highest expression level of FSHR, the expression level of Wnt4 was also significantly higher than the follicle with the lowest expression level of FSHR, with a log₂(fold change) of 2.12. We hypothesized that Wnt4 may play an important role in follicle selection and development, and therefore subsequently analyzed its function.

Wnt4 was expressed in both the prehierarchical and hierarchical follicles, but the expression level in the prehierarchical follicles was significantly higher than that in the hierarchical follicles (p < 0.01). The Wnt4 expression level was found to be highest in the small yellow follicles (6–8 mm), then declined significantly in the F5 follicles (p < 0.01), and this downward trend continued until F1, then slightly increased in the postovulatory follicles (POFs) (Figure 2A). On the protein level, Wnt4 was highly expressed in the prehierarchical, F5, and F3 follicles, but declined significantly in the F1 follicles and POFs (p < 0.01) (Figure 2B). In both the hierarchical and prehierarchical follicles, the Wnt4 mRNA and protein levels were significantly higher in the GCs than in the TCs (p < 0.01) (Figure 3).

To determine the effect of Wnt4 on steroidogenic enzyme gene expression, GCs were treated with Wnt4 overexpression vectors and shRNA. In pre-GCs, the expression levels of StAR (p < 0.01) and CYP11A1 (p ≤ 0.001) mRNA were significantly increased as a result of Wnt4 overexpression (Figure 4A). In GCs, the expression levels of StAR (p ≤ 0.001) and CYP11A1 (p ≤ 0.05) were also significantly increased (Figure 4B). The mRNA expression levels of StAR (p < 0.05) were significantly reduced in pre-GCs and GCs after knockdown of Wnt4 expression, whereas those of CYP11A1 were not significantly reduced (Figure 5).

As Wnt4 was highly expressed in GCs, we speculated that it is likely involved in GC proliferation in fast-growing follicles. Consequently, GCs were transfected with the overexpression vector pUSEamp-Wnt4 and the shRNA of Wnt4 to examine the effect of Wnt4 on their proliferation. We found that the overexpression of Wnt4 significantly stimulated the proliferation of GCs (p < 0.05) (Figure 6A). Conversely, when the Wnt4 expression level was knocked down, the proliferation of GCs significantly decreased (p < 0.01) (Figure 6B).

Follicle-stimulating hormone receptor, AMH, and OCLN are the key regulators of follicle selection, and so we determined the expression levels of these genes upon changing the expression level of Wnt4. In the prehierarchical GCs, the expression level of the mRNA for FSHR (p < 0.01) was significantly increased by the overexpression of Wnt4, and the expression levels of AMH (p < 0.05) and OCLN (p < 0.01) were simultaneously decreased (Figure 7A). Furthermore, the expression levels of AMH (p < 0.05) and OCLN (p < 0.01) mRNA were increased in prehierarchical follicle GCs treated with the shRNA of Wnt4, and the level of FSHR mRNA (p < 0.01) was simultaneously decreased (Figure 7B).

Follicle-stimulating hormone treatment at 5 ng/mL increased the expression of Wnt4 at both the mRNA (p < 0.05) and protein (p < 0.01) levels in the GCs of prehierarchical follicles (Figure 8A). In hierarchical follicles, with the increase of FSH concentration, the expression level of Wnt4 gradually increased (p < 0.01). Although a slight upward trend seems to occur, no significant difference of Wnt4 protein levels was observed in the hierarchical follicles (Figure 8B).

As the Wnt4 expression level changed during follicle development and was affected by the concentration of FSH, we further focused on the function of Wnt4 in follicular selection and development. In the GCs of prehierarchical follicles, FSH significantly increased the mRNA expression level of StAR and CYP11A1 (p < 0.01), although it had no significant stimulatory effect on progesterone and no effect on the expression of FSHR. When GCs were treated with a combination of FSH and Wnt4, Wnt4 suppressed the effect of FSH on the StAR and CYP11A1 mRNA levels (p < 0.01) (Figure 9A).

In the GCs of hierarchical follicles, FSH significantly increased the mRNA expression level of FSHR and the concentration of progesterone (p < 0.01), but not those of StAR and CYP11A1. When GCs were treated with a combination of FSH and Wnt4, progesterone secretion and steroidogenic gene expression were both significantly increased (p < 0.01) to levels exceeding those observed upon treatment with FSH or Wnt4 alone (Figure 9B).