2.1 Animals and Cell Culture
The animal study was reviewed and approved by the Experiment Center of Northwest A&F University (Approval No.2021028) and was in accordance with the Ethics on Animal Care guidelines for the use of animals in the experimental research. Performed under the control of the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No.398 set by the Ministry of Science and Technology, China. Patients were divided into repeat breeder and health groups according to OD620, leukocyte esterase (LE) and pregnancy outcomes 3 tests methods. Cow mastitis, ketosis, ovary inflammation and other diseases were excluded (n = 6). The dairy cows in the pregnant control group were at the time point of 45 days after delivery. The repeated breeders have experienced three or more pregnancy failures6,7.
The catheter was inserted into the tip of the uterus of repeat breeder and healthy dairy cows, respectively. The aseptic rinse solution (20 ml) was infused into the uterus, 10min was massaged through the cow's abdomen and then sucked out (n = 6). In order to minimize individual differences in animal responses, three repeat breeder sample pools from dairy cows and three sample pools from a control were constructed. Each sample pool consists of an equal volume (2.0 mL) of lavage solution, each sample pool consists of two cows, stored at -80°C until use. Centrifuge the collected samples at 2000 × g at 4°C for 10 minutes. And carefully remove the supernatant with a pipette8,9.
Primary bEECs were propagated in DMEM/F12 medium (Hyclone, Logan, UT, USA) supplemented with 10% FBS (ZETA, Lower Gwynedd Township, PA, USA) in a humidified atmosphere with 5% CO2 at 37 ℃. Cells are challenged by LPS (10 µg/mL) for 24 h10,11.
2.2 Preparation of leukocyte esterase (LE)
All operation steps are carried out according to the instructions of the kit (Fusheng Industrial, shanghai, China). Briefly, the standard is diluted by gradient. Blank holes, standard holes and sample holes to be tested are set respectively. Accurately add the standard sample 50 µL to the enzyme coated plate. The sample diluent 40 µL is first added to the sample hole, and then the sample 10 µL is added. The final dilution of the sample is 5 times. Add the sample to the bottom of the enzyme plate hole, shake and mix well. Incubate the sealing plate at 37°C for 30 min. The concentrated washing solution is diluted 30 times with distilled water. Remove the sealing film and discard the liquid. Fill each hole with detergent and leave it for 30 s before discarding. Repeat 5 times and pat dry. 50 µL of enzyme-labeled reagents were added to each hole, except for blank holes. Incubate the sealing plate at 37°C for 30 min again. Washing: Remove the sealing film and discard the liquid, fill each hole with detergent and leave it for 30 s before discarding, repeat 5 times and pat dry again. Add chromogenic agent A 50 µL and then chromogenic agent B 50 µL in each hole. Then 37°C avoid light and develop color for 10 min. The 50 µL of terminator was added to each hole to terminate the reaction. Zero with a blank hole, measurement of absorbance (OD value) of each hole at 450nm wavelength. The determination should be carried out within 15 min after the termination liquid is added. The repeat breeder is positive when OD value is greater than 55012,13,14.
2.3 Uterine lavage OD620nm
According to the existing literature, the 10% hypertonic saline solution was added to the uterine lavage sample at 1:1, mix evenly, Centrifuge at 4°C at 3000 × g for 30 min. The supernatant was collected and the equipartition sample of 0.2 mL was added to the 96-well microporous plate (SPAR, TECAN, Swiss). Optical density of uterine lavage samples measured at 620 nm. ULSOD620 greater than 0.07 is positive for repeat breeder15,16,17.
The pregnancy outcome of dairy cows diagnosed with repeat breeder group was judged to be failure. At the same time, the pregnancy outcome of the health group was judged to be successful. On the basis of the above test results, the target cattle were selected and then the proteomics was studied.
2.2 Preparation of colloidal particles
The sample was dissolved in a cleavage buffer I (7M urea, 2M thiourea, 4% propanesulfonic acid, 40 mM Tris-HCl, pH 8.5) containing a complete protease inhibitor. Centrifuge at 4 ℃ and 12000 × g for 10 minutes. The supernatant was detected by dioctanoic acid (BCA) protein analysis quantitative kit. All steps are carried out in accordance with the reagent instructions. The sample was thawed on ice and prepared for short-range sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12%) according to the manufacturer's instructions. The gel was stained with colloidal Coomassie blue. After gel staining, each channel was sliced and digested in the gel with SimplyBlue safe staining (Invitgen, USA). Unless otherwise stated, all steps are carried out at room temperature18,19.
2.3 Digestion in protein gel
A gel containing 100 µg of proteins was mixed with dithiothreitol (final concentration of 10 mM) and kept at 60°C for 70 min. After the sample was cooled, iodine acetamide with a final concentration of 66 mM was added and reacted away from light at room temperature for 40 minutes.
After that, 200 µL of 50 mM NH4HCO3 solution was added to the filter and centrifuged at 14000 × g for 25 min, repeat. Finally, 1.25µg of trypsin was added and the mixture incubated at 40°C overnight. The digestion procedure was stopped by adding formic acid (10%), and digested peptides were collected by centrifuging at 14000 × g for 15 min. After desalting with the C18 tips, the collected peptides were vacuum-dried. The Ziptip C18 micropipette tips (Millipore) were used to purify the peptides prior to adding 0.1% formic acid for LC-MS/MS analysis20,21.
2.4 LC–MS/MS Analysis
We used the Easy-nLC 1200 system coupled with a Orbitrap Fusion Lumos mass spectrometer for LC-MS/MS analysis, according to the method reported previously. Dried samples were re-suspended with mobile phase A (0.1% formic acid in water). Then, samples were loaded onto a pre-column (100 µm × 2 cm; 5 µm; C18 particles) and separated by linear gradient elution with mobile phase B on an analytical column at a flow rate of 300 µL/h. The following conditions were used: mobile phase A, 0.1% methanoic acid; mobile phase B, 0.1% methanoic acid and 80% ACN; flow rate, 300 nL/min; and analysis time, 0 min (10% phase B), 1 min (15%), 9 min (23%), 19 min (35%), 49 min (55%), 50 min (100%), and 60min (100% phase B). The MS conditions were as follows: First-order mass spectrometry parameters: Detector Type: Orbitrap, Resolution: 60000, Cycle time(sec): 3, RF lens (%): 30, AGC target: 4.0e5, Microscans:1, Data type: profile, Maximum IT: 50 ms and Scan range: 375–1500. Secondary mass spectrometry parameters: Detector Type: Orbitrap. Resolution: 15000, first mass (m/z): 110, AGC target: 5.0e4, Maximum IT: 256, microscans: 1, data type: centroid, HCD Collision Energy (%):3022,23,24.
2.5 Data processing
We analyzed all LC-MS/MS raw data files using Proteome Discoverer 2.2 software (version 2.2; Thermo Fisher Scientific). Difference screening criteria: Ratio > 1.5 or < 0.67, p-value < 0.05. We searched for data in a “Ruminantia” database from UniProt (protein database: uniprot-bovine-lzj20220113.fasta). Analysis settings: Enzyme Name, Trypsin (Full), Precursor Mass Tolerance, 20 ppm, Fragment Mass Tolerance, 0.5 Da, Static Modification: Carbamidomethyl/ + 57.021Da(C). Processing node 1. Spectrum Properties Filter: Lower RT Limit, 0. Upper RT Limit, 0. Min. Precursor Mass, 350 Da. Max. Precursor Mass, 5000 Da. A maximum of two missed cleavages was allowed. The maximum false discovery rate for peptide and protein was specified as 0.01, the LFQ (Label-Free Quantitation) was enabled with the LFQ minimum ratio count to 1. Regression Settings: Regression Model, Non-linear Regression, Parameter Tuning, Coarse. The other settings were kept as default. GraphPad Prism v7.0 was used with all data pre-tested for normality. And the values are presented as mean ± SD. Difference between health and repeat breeder groups was analyzed with Student's t-test or one-way ANOVA. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD041189. The dataset identifier of sub-project is IPX0006178001. Links: https://www.iprox.cn/page/SSV024.html;url=1683365319452QLaV. Passport: ZS7B25,26,27.
2.6. Bioinformatics
2.6.1 GO and KEGG annotations
The Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/orhttp://www.kegg.jp/) was used to predict the main metabolic pathways. The protein sequences of differentially expressed proteins were retrieved in batches from the UniProtKB database (http://www.uniport.org/, June 13, 2017, release) in FASTA format. Functional annotation was performed using the Gene Ontology (GO) database (http://www.geneontology.org) and included the cellular component, molecular function, and biological process. The GO mapping and annotation (http://geneontology.org/) results were plotted using Cluster 3.0 and Blast2 GO (version 3.3.5). The GO annotation results were plotted using R scripts. Retrieve the credibility is ≥ 95%, false positive is < 5% in the database; difference ratio of proteins is ≥ 1.5-fold and p-value is ≤ 0.05. The FASTA protein sequence of differentially expressed proteins (DEPs) were blasted against the online GO database (http://geneontology.org/) to retrieve their GO annotations and were subsequently mapped to pathways in KEGG. GO and KEGG pathway enrichment analyses were applied based on Fisher’s exact test. The Benjamini-Hochberg correction for multiple tests was further applied to adjust the derived p-values. Only functional categories and pathways with p-values under a threshold of 0.05 were considered significant. The analysis showed the 20 most relevant pathways sorted by p-value. The path-way enrichment analysis was done using database for annotation, visualization and integrated discovery28,29,30.
2.6.2 Hierarchical Clustering
The related protein data were used to perform hierarchical clustering analysis. For this purpose, Cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm), Java TreeView soft-ware (http://jtreeview.sourceforge.net) and R scripts were used. The Euclidean distance algorithm for similarity measurement and the average linkage clustering algorithm (clustering using the centroids of the observations) for clustering were selected during hierarchical clustering30,31,32.
2.6.3 Protein-Protein Interaction (PPI) Networks
The PPI information for the studied proteins using the gene symbols and STRING software. The PPI information for the studied proteins was retrieved from the IntAct molecular interaction database (http://www.ebi.ac.uk/intact/) using the gene symbols and STRING (http://string-db.org/) software. Difference ratio of proteins is ≥ 1.5-fold and p-value is ≤ 0.05, identified proteins must be redundancy by all artificial. The minimum interaction score was set to medium at 0.40 as the confidence score. The primary de-fault setting was used for the analysis. The results were downloaded in XGMML for-mat and imported into Cytoscape5 software (http://www.cytoscape.org/, version 3.2.1) to visualize and further analyze the functional PPI networks32,33,34.
2.7 The result of Elisa
After stimulation, the cells supernatant was collected for IL-6 and TNF-α detection using enzyme-linked immunosorbent assay kits (Solarbio, Beijing, China) at 24 h.
2.8 Western blot
Equivalent amounts of protein (15.0 µg) were loaded on 12% SDS-PAGE gels and then transferred onto 0.45µm nitrocellulose membrane (Millipore; Bedford, MA). The samples were incubated with anti-ERK (T-40071; Abmart; diluted 1:2000), anti-p-ERK (T-40072; Abmart; diluted 1:2000), anti-p-PI3K (T-40065; Abmart; diluted 1:2000), anti-p-JNK (T-40074; Abmart; diluted 1:2000) and anti-p-JUN (CY5897; Abways; diluted 1:2000). The anti-IMDH1(YT-6793; Immunoway; diluted 1:2000). The membranes were incubated for 2.5 h with horseshoe radish peroxidase-labeled secondary antibody (ZHONGSHANJINQIAO, China) at room temperature. Detection was performed (Dining, Beijing, China) and recorded by film exposure35,36.
2.9 Confocal microscopy
The cells were fixed by 4% paraformaldehyde for 20 min, then permeabilized with 0.5% triton-x diluted for 20 min. The cells were blocked with 10% FBS in 1× PBS containing 0.5% Tween-20. The cells were washed and added the secondary antibodies for 2 h. The cells were observed by a Nikon A1+/A1R + microscope (30 × objective, Nikon Inc, Melville, NY). The anti-ERK (T-40071; Abmart; diluted 1:200), anti-p-ERK (T-40072; Abmart; diluted 1:200), anti-FN1 (T-59537; Abmart; diluted 1:200) and anti-p-PI3K (T-40065; Abmart; diluted 1:200) were purchased from Abmart antibodies36,37.