Fifteen randomly sampled Hy-line brown hens were randomly divided into three biological groups, five hens in each group. These hens have been laying regularly for at least one month (28 weeks old, with mean body weight of 2.1± 0.12 kg) that were housed under standard conditions with free access to food and water and vaccination was performed according to recommendations from Hy-line International. From each hen, small yellow follicles 6–8 mm in diameter and hierarchical follicles 12–15 mm in diameter were separately collected, egg yolk was carefully squeezed out with tweezers, washed with phosphate buffered saline (Hyclone), immediately frozen in liquid nitrogen and used for transcriptomic and proteomic analyses. All sampled hens were killed by cervical dislocation immediately after oviposition. 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 for transcriptomic and proteomic analyses were prepar for total RNA and proteins.
Total RNA was isolated with TRIzol reagent (Invitrogen, UK) in accordance with the manufacturer’s instructions. Then 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 total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV 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, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). The library quality was assessed on the Agilent Bioanalyzer 2100 system (Agilent, USA). The library preparations were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated, and clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Gene expression level was quantified with featureCounts v1.5.0-p3 and FPKM.
Genes with an adjusted p-value <0.05 and |log2FoldChange| > 1 found by DESeq2 were assigned as differentially expressed. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathway enrichment analysis of differentially expressed genes were implemented by the clusterProfiler R package,and the P-value less than 0.05 were considered significantly enriched by differential expressed genes.
Sample processing and Liquid Chromaography Coupled with Tandem Mass Spectrometry (LC-MS/MS)
Proteins in the tissues were extracted with lysis buffer containing 8 M urea (Sigma) and 1% protease inhibitor cocktail (Calbiochem) and the protein concentration was determined with a BCA kit (Beyotime) according to the manufacturer’s instructions. Protein enzymolysis was performed using trypsin (Promega). After trypsin digestion, peptides were desalted on a Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the TMT kit (Thermo). The tryptic peptides were fractionated by high pH reverse-phase HPLC using an Agilent 300 Extend C 18 column (5 μm particles size, 4.6 mm ID, 250 mm length). The tryptic peptides were dissolved in 0.1% formic acid (Fluka) (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; the flow rate was constant at 400 nL/min; an EASY-nLC 1000 UPLC system was used. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in a Q ExactiveTM Plus spectrometer (Thermo) coupled online to the UPLC system. The electrospray voltage was 2.0 kV. The m/z scan range was 350 to 1800 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 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 followed by 20 MS/MS scans with 15.0 s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z.
The resulting MS/MS data were processed using the Maxquant search engine (v.22.214.171.124). 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. Mass tolerance for the precursor ions was set to 20 ppm in the first search and to 5 ppm in 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 oxidation of Met was specified as a variable modification. 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 enriched pathways. Differentially expressed proteins(DEPs) were identified with a threshold of 0.05 and fold change of > 1.5.
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; constant flow rate of 400 nL/min was maintained using an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) using a Q ExactiveTM Plus spectrometer (Thermo) connected to the UPLC system. The electrospray voltage was 2.0 kV. The m/z scan range was 350 to 1000 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 NCE set at 27, and the fragments were detected using an Orbitrap at a resolution of 17,500. A data-independent procedure alternated between an MS scan followed by 20 MS/MS scans. 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 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). Peptide settings: the enzyme was set as trypsin [KR/P]; the maximal missed cleavage 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. Transition settings: precursor charges were set as 2, 3; ion charges were set as 1, 2; and 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.
Real-time quantitative PCR
The total RNA was extracted from the S and F ovarian follicles that were used for proteome analysis using TRIzol reagent (Invitrogen). Synthesis of the cDNA was performed using 1 μg of the RNA by a PrimeScript RT reagent kit with a 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) as follows: 95°C for 30 s, 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 . The mRNAs of VLDLR, VLDLR1，WIF1, NGFR, AMH, BMP15, GDF6 and MMP13were quantitatively analyzed by this method. Quantification was accomplished by standardizing the reactions versus β-actin. All primers are listed in Additional file 4: Table S4.
Differences in mRNA expression between fillicles 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 of SAS (version 9.2). In one experiment, each treatment was repeated four times, and at least three independent experiments were performed. All data were presented as the mean ± SEM (n = 4). The GO and KEGG analysis were performed by Fisher’s t-test. P<0.05 was considered significantly different.