Porcine ovaries were obtained from pre-pubertal crossbred Landrace gilts at a local abattoir and transported to the laboratory within 2 h in saline supplemented with 75 mg/L of penicillin G potassium and 50 mg/L of streptomycin sulfate at 35–37°C. Follicular contents were aspirated from antral follicles (3–8 mm in diameter) using a disposable syringe with 18-gauge needle. The sediments containing cumulus oocyte complexes (COCs) were washed twice in Tyrode’s lactate-HEPES-polyvinyl alcohol (TLH-PVA) medium (Funahashi et al., 1997). COCs were selected under a stereomicroscope (Olympus, Tokyo, Japan), and those with dense cumulus cells (CCs) and uniform cytoplasm were used in the experiments.

In each experimental batch for oocyte collection, about three-fifths of the obtained COCs were vitrified, warmed, and then cultured for maturation, and the remaining COCs were directly subjected to IVM as the control. After IVM, mature metaphase II (MII) oocytes (first polar body extrusion) from these two groups were collected for the following proteomic experiments.

Porcine GV oocytes were subjected to vitrification and warming in the form of COCs, according to a previous report (Wu et al., 2017). All solutions were prepared using a base medium (BM), which was Dulbecco’s phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, United States) supplemented with 20% (v/v) synthetic serum substitute (Irvine Scientific, Santa Ana, CA, United States). First, COCs were put into BM for 3 min and then equilibrated with 5% (v/v) ethylene glycol (EG) for 10 min at 25°C. Subsequently, 10–15 COCs for each group were exposed to vitrification solution (VS) consisting of BM supplemented with 0.6 M sucrose, 50 mg/ml of polyvinylpyrrolidone and 35% (v/v) EG. After 20–30 s at 25°C, these COCs were loaded onto a Cryotop carrier (Kitazato Biopharma, Shizuoka, Japan) with minimum volume of VS and immediately plunged into LN₂.

Warming manipulation was performed on a 42°C hot plate, and warming solutions were also pre-heated to 42°C. For warming, the tip of Cryotop was dipped into 1.0 M of sucrose for 1 min. The vitrified COCs were picked out and transferred stepwise into 0.5 and 0.25 M of sucrose for 2.5 min, respectively. Finally, they were incubated in BM for 5 min and then submitted to IVM.

For IVM, about 50–70 COCs were cultured in each well of a 24-well plate (Costar, Corning, NY, United States) containing 500 μl of IVM medium covered by mineral oil for 42–44 h at 39°C in an atmosphere of 5% CO₂ with saturated humidity. The IVM medium was TCM-199 (Gibco, Grand Island, NY, United States) supplemented with 3.05 mM of D-glucose, 0.57 mM of cysteine, 0.91 mM of sodium pyruvate, 10% (v/v) porcine follicular fluid, 10 ng/ml of epidermal growth factor, and 0.5 μg/ml of each follicle-stimulating hormone and luteinizing hormone.

With regard to the vitrified COCs, their survival was evaluated after 2 h of IVM culture based on morphological characteristics under a stereomicroscope. Oocytes with disappeared vitelline membrane and/or altered cytoplasm were considered dead and removed, with surviving COCs continued to IVM.

At the end of IVM, both fresh and vitrified oocytes were gently denuded of CCs by repeated pipetting in TLH-PVA medium supplemented with 0.1% (w/v) hyaluronidase. Only MII oocytes with evenly granular cytoplasm were selected and then stored at –80°C to provisionally conserve. When the total number of collected oocytes was enough, they were pooled based on each sample requirement and then prepared for TMT and PRM analyses.

Three biological replicates were performed, and approximately 1,500 oocytes were used for each sample. For protein extraction, all samples were lysed with lysis buffer (8 M of urea, 1% Protease Inhibitor Cocktail) on ice using a high-intensity ultrasonic processor, in order to obtain the supernatant following centrifugation at 12,000 g at 4°C for 10 min. Protein concentration was determined using Bicinchoninic Acid Protein Assay Kit (Pierce, Rockford, IL, United States), according to the manufacturer’s instructions.

For digestion, protein solution was reduced with 5 mM of dithiothreitol (final concentration) for 30 min at 56°C, alkylated with 11 mM of iodoacetamide for 15 min in darkness at room temperature, and then diluted to urea concentration of less than 2 M. The trypsin at a mass ratio of 1:50 (trypsin:protein) at 37°C overnight was used to the first digestion and continued for a post-digestion with 1:100 mass ratio (trypsin:protein) for 4 h.

After trypsin digestion, peptides were desalted by Strata X C18 SPE column (Phenomenex, Torrance, CA, United States), vacuum-dried, and then reconstituted in 0.5 M of triethylammonium bicarbonate (TEAB). For TMT labeling, one unit of TMT reagent (Thermo Fisher Scientific) was thawed, reconstituted in acetonitrile, and mixed with peptides for 2 h at room temperature. Then the peptide mixtures were pooled, desalted, and dried by vacuum centrifugation.

High pH reversed-phase high-performance LC (HPLC) was performed to fractionate the labeled peptides, using an Agilent 300 Extend C18 column (5-μm particles, 4.6-mm i.d., 250-mm length; Agilent, Santa Clara, CA, United States). Briefly, tryptic peptides were first separated with a gradient of 8 to 32% acetonitrile (pH 9.0) over 60 min into 60 fractions, combined into nine fractions, and then vacuum-dried. Subsequently, the peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15-cm length, 75-μm i.d.) to elute with gradient solvent B (0.1% formic acid in 98% acetonitrile). A linear gradient of solvent B was used as follows: 7 to 16% over 50 min, 16 to 30% in 35 min, 30 to 80% in 2 min, and 80% for the last 3 min. Flow rate was 400 nl/min on an EASY-nLC 1000 ultraperformance LC (UPLC) system (Thermo Fisher Scientific, Waltham, MA, United States).

The peptides were subjected to nanospray ionization with a voltage of 2.0 kV and then detected by MS/MS in Q Exactive Plus (Thermo Fisher Scientific, Waltham, MA, United States) coupled online to the UPLC system. MS and MS/MS spectra were acquired in the Orbitrap with 60,000 resolution at 350–1,550 m/z and 30,000 resolution at 100 m/z, respectively. A data-dependent acquisition was performed with the following parameters: each MS scan followed by 20 MS/MS scans with 30.0-s dynamic exclusion. After MS scan, the 10 most abundant precursor ions were selected for higher-energy collisional dissociation (HCD) fragmentation with a normalized collision energy (NCE) setting of 32%. Automatic gain control (AGC) and maximum injection time (max IT) were set at 5E4 and 70 ms, respectively.

The resulting MS/MS data were processed using Maxquant search engine (v1.5.2.8) against the Sus scrofa UniProt proteome database (40,708 sequences) concatenated with reverse decoy database. The parameters were set as follows: (1) trypsin/P was specified as the cleavage enzyme; (2) two missing cleavages were allowed; (3) the minimum peptide length was seven amino acids; (4) the maximum number of modifications per peptide was 5; (5) the mass tolerance for precursor ions was 20 ppm in the first search and 5 ppm in the main search; (6) fragment ion mass tolerance was 0.02 Da; (7) carbamidomethylation on cysteine was fixed modification; (8) oxidation on methionine and N-terminal acetylation were variable modification; and (9) false discovery rate was adjusted to <1%. Student’s t-test was used to evaluate the significant differences. The proteins with fold change of ≥1.20 or ≤0.83 and p-value < 0.05 were considered as differentially expressed proteins (DEPs) on the basis of the related TMT or iTRAQ studies.

Protein Gene Ontology (GO) annotation was derived from the UniProt-GOA database¹ according to biological process, cellular component, and molecular function (Barrell et al., 2009). If proteins are not annotated by this database, the InterProScan² software was used to annotate GO function based on protein sequence alignment. The protein subcellular localization was predicted by Wolfpsort³ (Horton et al., 2007). We used an online service tool KAAS⁴ to annotate Kyoto Encyclopedia of Genes and Genomes (KEGG) database descriptions (Kanehisa et al., 2017). Subsequently, all proteins were mapped to corresponding pathways in the database using KEGG mapper⁵. As a result, a two-tailed Fisher’s exact test was employed to test the enrichment of the DEPs against all identified proteins. GO terms and KEGG pathways with a corrected p-value < 0.05 were considered to be significantly enriched. Protein–protein interaction (PPI) network was analyzed by STRING 11.0⁶ and then visualized in Cytoscape software (version 3.7.2).

Only proteins identified with high confidence peptide sequence were selected for PRM validation based on the TMT data. Three biological replicates were included, each of which was performed with around 1,000 oocytes. First, tryptic digested peptides through the TMT method described above were dissolved in solvent A and eluted using a homemade reversed-phase analytical column with gradient solvent B (6 to 25% over 40 min, 25 to 35% in 12 min, climbing to 80% in 4 min, and holding at 80% for the last 4 min) at 500 nl/min of flow rate. Then, the eluted peptides were analyzed using nanospray ionization source and Q Exactive Plus coupled online to the UPLC. An electrospray voltage of 2.2 kV was applied. By the Orbitrap, full MS was detected at a resolution of 70,000 with 350–1,060 m/z scan range (AGC, 3E6; max IT, 50 ms), followed by 20 MS/MS scans at a resolution of 17,500 (AGC, 1E5; max IT, 120 ms; isolation window, 1.6 m/z) in a data-independent acquisition. Precursor ions were fragmented through HCD with an NCE of 27. The PRM data were processed using Skyline software (version 3.6, MacCoss Lab, University of Washington, United States) (MacLean et al., 2010). The results for each peptide were quantified according to the fragment ion peak area from its corresponding transitions, and statistical significance was set at p-value < 0.05 using Student’s t-test.

In the present study, the survival rates of vitrified GV oocytes after both 2 h of warming and IVM were 86.8 and 83.1%, respectively; the fresh GV oocytes had 93.5% survival following IVM. Moreover, the MII rate was 85.9% for vitrified oocytes and 88.6% for fresh oocytes.

First of all, biological replicates were validated by the relative standard deviation distribution, which displayed the precision and reproducibility of our proteomic datasets (Supplementary Figure 1A). By a strict quality control, we obtained a total of 3,51,303 spectra (58,348 matched), and 27,619 peptides (26,173 unique peptides) were detected from among them (Figure 1A). Moreover, the average mass error of peptides was less than 6 ppm, indicating a high mass accuracy of the MS data as requirement during the processes (Supplementary Figure 1B). Further analysis showed that the lengths of most peptides were distributed at the range of 7 to 20 amino acids, which meant reliable results (Supplementary Figure 1C). Finally, 4,499 proteins were identified, 3,823 of which were quantified (Figure 1A). The detailed protein information is provided in Supplementary Table 1.

Following statistical analysis, 153 proteins with fold-change ≥1.20 or ≤0.83 and p-value < 0.05 were considered as the DEPs. Among them, a total of 94 up-regulated and 59 down-regulated DEPs were identified (Supplementary Table 2). In addition, a heatmap represented a hierarchical cluster of the DEPs, and a volcano plot also indicated average changes of individual protein abundance (Figures 1B,C).

According to subcellular localization predictions, these DEPs were mainly distributed in the “cytoplasm” (27%), “extracellular” (27%), “nucleus” (25%), “plasma membrane” (6%), and “mitochondria” (6%) (Figure 2A and Supplementary Table 3). Next, GO functional classification was performed for the DEPs. We found that these DEPs were cataloged in 27 GO terms, including 12 biological process, 8 cellular component, and 7 molecular function (Figures 2B–D and Supplementary Table 4). More detail, the top five GO terms in biological process consisted of “cellular process” (17%), “biological regulation” (15%), “metabolic process” (13%), “single-organism process” (13%), and “response to stimulus” (9%). The cellular component results showed that “cell” (27%), “organelle” (22%), “extracellular region” (16%), “macromolecular complex” (13%), “membrane” (11%), “membrane-enclosed lumen” (7%), and “cell junction” (2%) were mostly classifications. Moreover, “binding” (63%) and “catalytic activity” (18%) were the two most prominent terms in the molecular function. On the other hand, the functional classification of DEPs was also predicted by performing Clusters of Orthologous Groups of protein/EuKaryotic Orthologous Groups (COG/KOG) analysis. As shown in Figure 3 and Supplementary Table 5, these DEPs were assigned to 20 COG/KOG categories, and the largest category was “signal transduction mechanisms,” followed by “transcription,” “general function prediction only,” “post-translational modification, protein turnover, chaperones,” “defense mechanisms,” and so on.

To further predict the possible roles of DEPs, functional enrichment analysis was conducted according to GO annotation and KEGG pathway. First, detailed information of GO term enrichment is shown in Figure 4 and Supplementary Table 6. Within the cellular component, the enriched terms were “extracellular space,” “membrane attack complex,” “pore complex,” “extracellular region,” etc. Molecular function enrichment indicated peptidase and endopeptidase inhibitor/regulator activity. Regarding biological process, the top five terms included “protein activation cascade,” “activation of immune response,” “negative regulation of hydrolase activity,” “positive regulation of immune response,” and “negative regulation of cellular metabolic process.” In addition, the KEGG enrichment analysis found several main pathways such as “complement and coagulation cascades,” “thyroid hormone synthesis,” and “spliceosome” (Figure 4 and Supplementary Table 7).

For elucidating the functional interactions of DEPs, we performed a PPI network analysis among DEPs. As shown in Figure 5, three highly interconnected clusters were identified in this PPI network. There were seven DEPs with a high degree of connectivity, including plasminogen (PLG, P06867), complement C5a anaphylatoxin (C5, A0A286ZKB4), complement component C9 precursor (C9, F1SMJ6), serum albumin (ALB, A0A287AMK0), complement component C8 beta chain precursor (C8B, A0A287AT36), complement component C8 gamma chain precursor (C8G, A0SEH3), and complement C8 alpha chain (C8A, F1S788); and most of them participated in complement component. In addition, pleiotropic regulator 1 (PLRG1, F1RX38), thioredoxin like 4A (TXNL4A, A0A287A0V4), U4/U6.U5 tri-snRNP-associated protein 1 (SART1, F1RU31), small nuclear ribonucleoprotein polypeptide A’ (SNRPA1, F1RWS8), and splicing factor 3a subunit 3 (SF3A3, F1SV40) constructed an interconnected cluster and were involved in the “spliceosome” pathway.

To validate the TMT results, a PRM analysis was performed to detect the expression of 10 candidate DEPs. Due to the requirements of protein characteristics and abundance, we obtained the abundant values of seven proteins by quantitative data of target peptide fragments, including ATP synthase subunit e (ATP5ME, Q9MYT8), calcitonin receptor-stimulating peptide 3 (CRSP3, A0A286ZNZ6), tropomyosin alpha-4 chain (TPM4, P67937), vimentin (VIM, P02543), bone morphogenetic protein 15 (BMP15, F1RV73), complement C3 (C3, I3LTB8), and serum albumin (ALB, A0A287AMK0). As shown in Figure 6, these proteins were exactly the same trend as quantified using TMT method, although the fold change varied between the two techniques. Besides BMP15, they showed significant differences between groups.