All ovary buffalo specimens (Bubalus bubalis) were acquired from a local commercial slaughterhouse. Two 3.8–4.0 g ovaries were harvested from female buffalo (age 4.0–6.0 years) in the corpus hemorrhagicum (CH) phase, two 4.1–4.5 g ovaries were harvested from female buffalo (age 4.5–5.5 years) in the corpus luteum (CL) phase, and two 3.3–3.9 g ovaries were harvested from female buffalo (age 5.0–6.5 years) in the corpus fibrosum (CF) stage. In the months of September and October of the same year, all ovaries were collected. Detailed ovary sample data was shown in Additional File 5: Table S5 and Additional File 6: Figure S1. Figure 5 shows the workflow of these studies. Six ovaries from three phases (CH, CL, CF) were used for sample preparing, and the six ovaries were split into two distinct samples for two biological replicates. The ovaries were transported in precooled physiological saline to the laboratory. The lysis buffer (7 M urea, 3 per cent CHAPS, 1.5 M thiourea, 1 per cent DTT, and 1 per cent v / v protease inhibitor cocktail) was used to remove the ovarian protein after liquid nitrogen grinding. After incubation with vortexing every 15 min for 60 min on ice, the samples were centrifuged at 4 ° C at 12,000 / g for 30 min, and the supernatants of lysates were collected for the next experiment. The Bradford assay kit quantified the levels of the extracted proteins.
Digestion of Protein and TMT-labeling
One hundred micrograms of protein from each sample was precipitated overnight in the precooled acetone at −20 ° C and then centrifuged at 13,000 g for 30 min at 4 ° C. The supernatant was removed, and precipitation was dissolved in a combination of 100 mM TEAB (triethylammonium bicarbonate), 5 uL 2 percent SDS, and 55 uL of ultrapure water. The protein reduction alkylation was performed at 55 ° C with the 5 uL of 200 mM trichloroethyl phosphate for 60 min, and then 5uL of 375 mM iodoacetamide was added in the dark and incubated at room temperature for 30 min. The sample was then precipitated for 3 hours in cold acetone (1:6) at −20 ° C and then placed in 100 uL of 100 mM triethyl ammonium bicarbonate. Finally, for 16-18 hours, each sample was digested with 2.5 uL of trypsin (1 ug / uL) at 37 ° C. The TMT reagents were used to label the peptides in accordance with the manufacturer's instructions. Briefly, 41 uL of anhydrous acetonitrile was added to each TMT label tube (0.8 mg), and each sample was marked by adding 20 uL of TMT label and incubated at room temperature for 60 min. After that, 8 uL of 5 per cent hydroxylamine was added to each sample and incubated for 15 min to end the reaction. Samples of CH, CL, and CF buffalo ovaries were marked with 126, 127, and 130, respectively, for the first biological replicate; samples of CH, CL, and CF buffalo ovaries were marked with 128, 129, and 131, respectively, for the second biological replicate. The six samples with six tags were blended by equal quantity before downstream separation after labelling.
High pH Reverse Phase Separation and Nano-LC-MS/MS Analysis
Each sample was evaporated in a vacuum and resuspended in 50 μL buffer A (98% ddH2O, 2% acetonitrile; pH 10.0) and separated by high-pH reversed-phase liquid chromatography (RP-HPLC) column (2.1 ± 100 mm, three μm, 150 Å, C18). We used buffer A and buffer B (98 per cent acetonitrile, 2 per cent ddH20; pH 10.0) for a 60-min linear gradient (4 per cent –20 per cent buffer B for 30 min, 20 percent –95 percent buffer B for 25 min, and 95 percent buffer B for 5 min). The fractions eluted from the column were collected every 1.5 min, and according to the maximum intensity, 20 fractions were gathered. The gathered fractions were desalted using a column of ZipTips C18 (Millipore, Billerica, MA, USA). The fractions were evaporated with a vacuum after desalination, and 10 uL of solvent A (2 per cent acetonitrile and 0.1 per cent formic acid) was used to resuspend the samples. In a trap column (PepMap RSLC C18 column, 50 um15 cm, 2 um nanoViper, Thermo Fisher Scientific, Bremen, Germany), a volume of 2 μl from each sample was loaded, and a maximum pressure (600 bar) was applied and a column for analysis (Acclaim ® PepMap100C18 column, 0.075 about 150 mm, 3μm, 100Å, Thermo Fisher Scientific, Bremen, Germany) was used for elution at a rate of 300 nL / min. Next, we used buffer A (2 percent acetonitrile and 0.1 percent formic acid) and buffer B (98 percent acetonitrile and 0.1 percent formic acid), followed by a gradient of 60 min (5 percent –40 percent buffer B for 45 min, 40 percent –100 percent buffer B for 10 min, 100 percent buffer B for 5 min) to separate peptides. Finally, an LTQ-Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) linked to an Easy-nLC 1,000 nano fluid chromatography scheme (Thermo Fisher Scientific, Odense, Denmark) was used to analyze all peptide fractions online.
For mass spectrometry (MS) analysis, a data-dependent mode in the scan range of 350–1,800 m / z was performed, and the survey scans were captured by the Orbitrap analyzer at a mass resolution of 60,000 at 400 m / z. In the linear ion trap, under the mode of high-energy collision dissociation, 10 of the most intense precursor ions were selected for secondary mass spectrometry analysis (MS2). The dynamic exclusion parameter included a two exclusion count and a 40 s exclusion moment. Siloxane ions have been used for inner calibration (m / z = 445.1200).
Proteomic Data Analysis and Bioinformatics
The raw information files were processed and quantified using Proteome Discoverer v188.8.131.529 (Thermo Fisher Scientific, Massachusetts, USA) and searched using the SEQUEST algorithm against the UniProt Bos Taurus protein database (UP000009136, 24078 sequences, release 2017_03). The search parameters used were as follows: fixed changes, including cysteine carbamidomethylation (+ 57.02146 Da); TMT reagent adducts (+ 229.162932 Da) on lysine and amino peptide termini; and variable changes, including methionine oxidation (+ 15.99492 Da). Precursor ion mass tolerance was 20 ppm, and fragment ions were set to ± 0.5 Da, enzyme specificity, trypsin. The false discovery rate (FDR) was calculated using the peptide validator and based on a search for the decoy database. A p-value < 0.05 and a fold change > 1.5 were regarded important.
The GO (gene ontology) was used to annotate the recognized proteins, which consisted of BP (biological processes), CC (cellular elements) and MF (molecular functions), and the analysis was based on the UniProt-GOA database (http:/www.ebi.ac.uk/GOA/). The pathway analysis of differently expressed proteins was based on the KEGG (Encyclopedia of Genes and Genomes) database. The online KEGG Automatic Annotation Server (KAAS) service instruments were used to annotate the KEGG database description for each protein and to display the annotation outcomes using other online KEGG service instruments and the KEGG mapper. The interaction between groups of differential expression protein (DEPs) was extracted from the database of Search Tools for the Retrieval of Interacting Genes / Proteins (STRING). A high trust (0.7) was selected to draw the protein-protein interaction map using Cytoscape 3.2.1 for the necessary interaction score and the active interaction sources, including text mining, tests, and databases.
The IDs conversion of identified proteins and homologous BLAST cross-species
All buffalo protein IDs were transformed to Uniprot database mouse (Mus musculus) IDs and human (Homo sapiens) IDs using blast software (Blastall v2.2.26). The program name (comparison technique) is blastp, and the screening criteria is the expectation value < 1e-5. A similarity comparison of cross-species (buffalo, mouse and human) was also performed with the amino acid sequences of DEPs found in buffalo. In addition, the analysis of the GO and KEGG pathway was used to annotate the converted IDs from DEPs, including mouse and human.
Verification of Differentially Expressed Proteins Using Quantitative qRT-PCR
Total RNA obtained from ovaries at separate phases (CH, CL, and CF, n=3) was performed by Trizol regeant (Invitrogen). To reverse-transcribe and collect the cDNAs, a Takara RNA polymerase chain response (PCR) kit was used. A Light Cycler 96 scheme (Roche, Switzerland) performed quantitative inverse transcription PCR (qRT-PCR) analyzes. The reaction solution (20 μL) is a composition of 1.0 μL of cDNA, ten μL of SYBR ® Premix Ex TaqTM II (Takara), 2 uL of primer mix (2 nM), and 7.0 μL of ddH2O. The 2-ΔΔCT technique was used to calculate the relative expression concentrations of targeted genes. Each gene was evaluated using different oocyte sets and tests were carried in three replicates. The primer sequences used forqRT-PCR analyses are provided in Additional File 4: Table S4.
Verification of Differentially Expressed Proteins Using Western Blotting
A western blot assay (n=3) evaluated the protein expression concentrations of PLK1, PGP, and HGS. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) was used to separate equal amounts of proteins from each sample (CH, CL and CF), and these samples were then transferred via a semidry western blot system (Trans-Blot ® TurboTM System, Bio-Rad, Singapore) to a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked for two h in 5 per cent nonfat milk at 37 ° C and incubated for 16-18 hours at 4 ° C with main antibiotics (1:1,000–5,000 dilution). Membranes were washed three times using TBST buffer (a mixture of TBS[ Tris-buffered saline] and Tween 20) and then incubated for two h at 37 ° C with secondary antibodies in the TBST buffer. Bands were visualized using an alkaline phosphatase detection kit (C3206, Beyotime Biotechnology Inc., Shanghai, China). The following commercially accessible antibodies were used as primary antibodies: anti-PGP9.5 mouse monoclonal antibody (ab72911 ; Abcam, Cambridge, MA, USA), anti-HGS rabbit polyclonal antibody (ab72053 ; Abcam), anti-actin (loading control) rabbit polyclonal antibody (bs-17654R ; Bioss biotechnology Inc., Beijing, China) and anti-Phospho-PLK1 (Thr210) rabbit polyclonal antibody (bs-3344R ; Bioss Biotechnology Inc., Beijing, China)
IHC was conducted on ovaries fixed with a polyoxymethylene (n=3). The ovaries embedded in paraffin were dewaxed and dehydrated, and 30 minutes of endogenous peroxidase activity was quenched with methanol comprising 3% H2O2. The paraffin parts were subjected to antigen extraction using microwave heating in the 0.01 M sodium citrate buffer (pH 6.0). These sections were blocked at room temperature for 2 hours using the 5 per cent BSA and then incubated overnight at 4 ° C with primary antibodies against PLK1, PGP, and HGS. The parts were then cleaned for 15min by PBS-TWeen-20, and this step was repeated three times. The parts were then incubated with secondary HRP-conjugated antibodies (CWBIO). The immunoreactive sites were visualized in brown after staining with diaminobenzidine (CWBIO), and the parts were stained with hematoxyl and assembled for observation using a bright-field microscope (Olympus, Japan). Instead of a primary antibody, negative checks were incubated with ordinary IgG.
IBM SPSS Statistics version 17.0 and GraphPad Prism version 5.0 were used for statistical analysis. The research information was displayed as mean ± standard deviation (SD). A student t-test was used to compare the qRT-PCR outcomes of two groups (p < 0.05 was considered statistically significant).