Tissue collection
Fifteen randomly sampled Hy-line brown hens from the same batch were divided into three biological groups, with five hens in each group. These hens had been laying regularly for at least one month (28 weeks old, with a mean body weight of 2.1± 0.12 kg) and were housed under standard conditions with free access to food and water. Vaccination was performed according to the recommendations from Hy-line International. The laying time of the sampled hens were recorded and the hens were killed by cervical dislocation after approximately ten hours after laying. From each hen, small yellow follicles 6-8 mm in diameter and hierarchical follicles 12-15 mm in diameter were separately collected, and the egg yolk was carefully squeezed out with tweezers, washed with phosphate-buffered saline (Thermo Fischer Scientific, MA, USA), immediately frozen in liquid nitrogen and used for transcriptomic and proteomic analyses. The Institutional Animal Care and Use Ethics Committee of Shandong Agricultural University reviewed and approved all procedures described in this study. This study was performed according to the Guidelines for Experimental Animals of the Ministry of Science and Technology of China. Three biological replicates were prepared for the transcriptomic and proteomic analyses of total RNA and proteins.
Transcriptome analysis
Total RNA was isolated with TRIzol reagent (Invitrogen, CA, USA) in accordance with the manufacturer’s instructions. Then, the RNA purity and integrity were checked using the NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA), respectively. Subsequently, mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations at an elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). The first-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3’ ends of the DNA fragments, NEBNext Adaptors with hairpin loop structures were ligated to prepare for hybridization. To select the cDNA fragments that were 250~300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). The library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent, CA, USA). The library preparations were sequenced on an Illumina Novaseq platform, and 150-bp paired-end reads were generated; the clean data (clean reads) were obtained by removing reads containing adapters and poly-N and low-quality reads from the raw data. The index of the reference genome was built and the paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5 [40]. The gene expression levels were quantified with featureCounts v1.5.0-p3 and FPKM [41].
Genes with an adjusted P-value <0.05 and a |log2FoldChange| > 1 according to DESeq2[42] were considered differentially expressed. Gene ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis of the differentially expressed genes were implemented by the cluster Profiler R package, and genes with P-values less than 0.05 were considered significantly enriched as differentially expressed genes [43].
Sample processing and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)
The proteins in the tissues were extracted with lysis buffer containing 8 M urea (Sigma Aldrich, MO, USA) and 1% protease inhibitor cocktail (Merck Millipore, MA, USA), and the protein concentration was determined with a BCA kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Protein enzymolysis was performed using trypsin (Promega, WI, USA). After trypsin digestion, the peptides were desalted on a Strata X C18 SPE column (Phenomenex, CA, USA) and vacuum-dried. The peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the TMT kit (Thermo Fischer Scientific, MA, USA). The tryptic peptides were fractionated by high-pH reverse-phase HPLC using an Agilent 300 Extend C 18 column (5 μm particle size, 4.6 mm ID, 250 mm length). The tryptic peptides were dissolved in 0.1% formic acid (Sigma Aldrich, MO, USA) (solvent A) and directly loaded onto an in-house-packed reversed-phase analytical column (150 mm length, 75 μm i.d.). The gradient included the following steps: from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, from 23% to 35% solvent B over 8 min and from 35% to 80% over 3 min; then, the column was eluted with 80% solvent B for the last 3 min of the run. The flow rate was constant at 400 nL/min, and an EASY-nLC 1000 UPLC system was used. The peptides were exposed to an NSI source followed by tandem mass spectrometry (MS/MS) with a Q ExactiveTM Plus spectrometer (Thermo Fischer Scientific, MA, USA) coupled online to the UPLC system. The electrospray voltage was 2.0 kV. The m/z scan range was 350 to 1,800 for the full scan, and the intact peptides were detected using an Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using an NCE set at 28, and the fragments were detected using an Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between an MS scan and 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The fixed first mass was set as 100 m/z.
The resulting MS/MS data were processed using the Maxquant search engine (v.1.5.2.8). The tandem mass spectra were searched against the Gallus gallus database (http://www.uniprot.org/proteomes/UP000000539) concatenated with the reverse decoy database. Trypsin/P was specified as the cleavage enzyme, and up to 2 missing cleavages were allowed. The mass tolerance for the precursor ions was set to 20 ppm for the first search and to 5 ppm for the main search, and the mass tolerance for the fragment ions was set to 0.02 Da. Carbamidomethyl was specified as a fixed modification of Cys, and the oxidation of Met was specified as a variable modification. The FDR was < 1%, and the minimum score for the peptides was > 40.
The GO proteome was derived from the UniProt-GOA database (www.http://www.ebi. ac.uk/GOA/). The Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to identify the enriched pathways. Proteins with a threshold of P<0.05 and a fold change of > 1.5 or <1/1.5 were identified as differentially expressed proteins (DEPs) between SY and F6 follicles.
Parallel reaction monitoring
Protein extraction and trypsin digestion were performed as described above. Then, the tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto an in-house-packed reversed-phase analytical column (150 mm length, 75 μm i.d.). The gradient included the following steps: from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 38 min, from 23% to 35% solvent B over 14 min, from 35% to 80% solvent B over 4 min, and 80% solvent B for 4 min; a constant flow rate of 400 nL/min was maintained using an EASY-nLC 1000 UPLC system. The peptides were exposed to an NSI source followed by tandem mass spectrometry (MS/MS) using a Q ExactiveTM Plus spectrometer (Thermo Fischer Scientific, MA, USA) connected to the UPLC system. The electrospray voltage was 2.0 kV. The m/z scan range was 350 to 1,000 for the full scan, and intact peptides were detected using an Orbitrap at a resolution of 35,000. Then, the peptides were selected for MS/MS using an NCE set at 27, and the fragments were detected using an Orbitrap at a resolution of 17,500. The data-independent procedure alternated between an MS scan followed by 20 MS/MS scans. The automatic gain control (AGC) was set at 3E6 for the full MS and at 1E5 for MS/MS. The maximal IT was set at 20 ms for the full MS and was switched to automatic mode for MS/MS. The isolation window for MS/MS was set to 2.0 m/z.
The resulting MS/MS data were processed using Skyline (v.3.6). The following peptide settings were used: the enzyme was set as trypsin [KR/P]; the maximal number of missed cleavages was set as 2; the peptide length was set as 8-25; variable modifications were set as carbamidomethylation of Cys and oxidation of Met; and the maximal variable modification was set as 3. The following transition settings were used: The precursor charges were set as 2, 3; the ion charges were set as 1, 2; and the ion types were set as b, y, p. The product ions were derived from ion 3 to the last ion, and the ion match tolerance was set to 0.02 Da.
Cell culture and cell treatment
All of the small yellow follicles and the hierarchical follicles were isolated from hens and placed in PBS. The small yellow follicles were treated with 0.1% collagenase II (MP Biomedicals, Santa Ana, CA, USA) at 38°C for 15 min to obtain the GCs. The yolk of the follicles was 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-BRL, NY, USA) at 38°C for 15 min with gentle oscillation in a centrifuge tube. After centrifugation, the GCs were suspended in culture medium that contained M199 (Gibco-BRL, NY, USA), 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel) and 1% penicillin/streptomycin (Solarbio, Beijing, China) and subsequently seeded in 24-well culture plates at a density of 1 × 105 cells/well. The number of viable cells (90%) was estimated using Trypan blue staining. The cells were cultured at 38°C in a water-saturated atmosphere of 95% air and 5% CO2. After 24 h, the cultured cells were subsequently treated with different concentrations (0, 5, 10, and 50 ng/ml) of recombinant FSH (R&D Systems, MN, USA). All the treated cells were collected after another 24 h for RNA extraction and qRT-PCR analysis.
Real-time quantitative PCR
Total RNA was extracted from the S and F ovarian follicles that were used for proteome analysis using TRIzol reagent (InvivoGen, CA, USA). Synthesis of the cDNA was performed using 1 μg of the RNA by using a PrimeScript RT reagent kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol. Real-time quantitative PCR was performed using a SYBR Premix Ex TaqTM II kit (TaKaRa, Dalian, China) on a Light Cycler 480 real-time PCR system (Roche, Basel, Switzerland) as follows: 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s and annealing and extension at 58°C for 20 s. The melting curves were obtained, and quantitative analysis of the data was performed using the 2−ΔΔCT relative quantification method [44]. The mRNAs of VLDLR, VLDLR1, WIF1, NGFR, AMH, BMP15, GDF6 and MMP13 were quantitatively analyzed by this method. Quantification was performed by standardizing the reaction results versus those of β-actin. All primers are listed in Table S5.
Statistical analysis
Differences in mRNA expression between follicles and follicular cells were evaluated by one-way ANOVA followed by Duncan’s multiple range test (P < 0.05) using the General Linear Model procedure in SAS (version 9.2). For one experiment, each treatment was repeated four times, and at least three independent experiments were performed. All data are presented as the mean ± SEM (n = 4). The GO and KEGG analyses were performed by using Fisher’s t-test. P<0.05 was considered to indicate a significant difference.