Mouse monoclonal anti-α-tubulin-FITC antibody was purchased from Sigma (St. Louis, MO, USA). Rabbit polyclonal anti-m6A (202003) was purchased from Synaptic Systems (Goettingen, Germany); Rabbit H3K9me3 (ab8898) and H3K4me3 (ab8580) were purchased from Abcam (Cambridge, MA, USA); Rabbit polyclonal anti-histone H4 (acetyl K16) antibody (ab109463) was purchased from Abcam; Rabbit monoclonal anti-γH2A.X antibody were purchased from Cell Signaling Technology (Danvers, MA, USA); Alexa Fluora 488 goat anti‐mouse (A-11001) and Alexa Fluora 594 goat anti-rabbit (A-11037) were purchased from Invitrogen (Invitrogen, MA, USA).
The collection and in vitro maturation (IVM) of porcine oocytes
The porcine ovaries were collected immediately after being slaughtered in a local abattoir, and then were transported to the laboratory within 2 hours in 0.9% NaCl supplemented with 5% penicillin-streptomycin at 35-36.5 °C. COCs (cumulus-oocyte complexes) were collected using a 10 ml syringe from 2–6 mm ovarian follicles. Then the COCs with more than three layers of cumulus cells were selected and cultured in the in vitro maturation (IVM) medium. A group of 200 COCs was cultured in 1 mL of maturation medium I (TCM-199 supplemented with 26 mM sodium bicarbonate, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.5 µg/mL follicle-stimulating hormone (FSH), 0.5 µg/ml luteinizing hormone (LH), 10 ng/ml epidermal growth factor (EGF), 0.1% PVA, 0.03% BSA and 0.1% penicillin/streptomycin) covered with liquid paraffin oil at 38.5 °C with 5% CO2 for 22–24 h, and then cultured in maturation medium II (the same medium with medium I without hormone) until 42–44 h according to our previous description .
The isolation and culture of porcine cumulus cells (CCs)
The CCs were isolated from COCs with 0.2% hyaluronidase, collected by a 15 mL centrifuge tube. After centrifuging at1500 g for 5 min, CCs were resuspended with DMEM/F12 medium (Gibco, Beijing, China), and subsequently plated onto 60-mm culture plate in DMEM/F12 supplemented with 10% FBS (Gibco, Beijing, China), 100 units/mL penicillin, and 100 µg/mL streptomycin in a humidified atmosphere with 5% (v/v) CO2 at 37 °C.
Cycloleucine (CL) treatment
In the present study, COCs were cultured in IVM medium supplemented with cycloleucine (CL) (A48105, Sigma) at a concentration of 10, 20, 40 mM, respectively.
Oocytes at Metaphase I (MI) and Metaphase II (MII) stages were collected at 27 h and 42 h, respectively. For cumulus cell (CCs) treatment, CCs were isolated from COCs by 0.2% hyaluronidase and then were treated with CL at a working concentration of 20 mM.
Calculation of area of CCs expansion and viability
Cumulus expansion was measured during the COCs in vitro maturation period, as described previously . Briefly, 40 COCs of each group were taken out of the incubators and captured by a fluorescence microscope. The size of each COC within different groups were measured at 0 h, 24 h and 44 h of in vitro maturation. The ImageJ software was used to calculate the area of CCs expansion according with the description previously. The cumulus expansion rate of control group was calculated as 100% at 44 h of in vitro maturation, and it was performed at least three times. For the evaluation of cell viability, COCs were digested with 0.2% hyaluronidase . After separating CCs with oocytes, the CCs were staining 0.4% trypan blue solution. Simultaneously, the cell viability was assessed by a light microscope (Nikon, Tokyo, Japan), living cells could not be stained with trypan blue. For oocytes survival rate, oocytes which possess intact zona pellucida and plasma membrane, evenly distributed cytoplasm were regarded as morphological survival. The cell viability was calculated as the percentage of viable cells out of total cells number and it was performed at least three independent replicates.
Parthenogenetic activation (PA) of porcine oocytes
Parthenogenetic activation of porcine oocytes was performed according with the description previously . Briefly, after digestion with 0.2% hyaluronidase for 5 min, denuded MII oocytes (with the first polar) were equilibrated in the activation medium (0.28 M mannitol, 0.5 mM Hepes, 0.1 mM MgCl2, and 1 mM CaCl2) for almost 20 seconds. And then the oocytes were activated by an ECM2001 electro-fusion instrument (ECM2001, BTX, USA) with two direct-current pulses of 1.2 kV/cm for 30 µs. Then the oocytes were cultured in PZM-3 medium (108 mM NaCl, 10 mM KCl, 0.35 mM KH2PO4, 0.4 mM MgSO4.7H2O, 25.07 mM NaHCO3, 0.2 mM Na-pyruvate and 2 mM Ca-lactate.5H20, 1 mM L-glutamine, 5 mM hypotaurine, 0.3% BSA, 20 mL/L BME amino acid and 10 mL/L MEM non-essential amino acid)) at 38.5 °C with 5% CO2 for 7 days. The cleavage and blastocyst rates of embryos were visually checked at 48 h and 7 days after PA.
Immunofluorescence (IF) staining
Immunofluorescence staining was performed according with our description previously . Briefly, the oocytes or embryos were removed of zona pellucida with 0.5% acidic Tyrode solution. After washed with PBS/PVA (1 mg/1 mL) for three times, oocytes or embryos were fixed with 4% paraformaldehyde (PFA) for 40 min at room temperature. The samples were permeabilized with 1% Triton-X 100/PBS for 20 min and blocked with 2% BSA/PBS for 1 h at RT. Then the samples were incubated overnight at 4 °C with primary antibodies according to manufacturer’s recommended dilutions. After being washed with PBS/PVA for three times, samples were stained with secondary antibodies in the dark for 2 h at 37 °C. Then the DNA was stained with 10 µg/mL DAPI for 15 min. The oocytes or embryos were mounted on coverslips in ProLong Diamond Antifade Mountant reagent (Life Technologies, USA). A fluorescence microscope (Nikon, Tokyo, Japan) was used to capture fluorescent images by setting the same parameters for all groups. The fluorescent images for α-tubulin were taken by a confocal laser-scanning microscope (LSM8800, Zeiss, Oberkochen, Germany). The relative fluorescence intensity was analyzed by Image J software (National Institutes of Health, Bethesda, MD) according to the previous description.
Cell number counting of blastocyst
Total cell numbers of blastocysts were assessed and calculated by staining with Hoechst33342 (10 µg/mL). Briefly, blastocysts were fixed with 4% PFA in PBS/PVA for 40 min, followed by permeabilization in 1% Triton-X 100/PBS for 30 min at RT. After being washed with PBS/PVA for 3 times (5 min/time), the blastocysts were stained with Hoechst33342 for 15 min at RT. Then the blastocysts were mounted on coverslips in ProLong Diamond Antifade Mountant reagent (Life Technologies, USA), and total cell number was observed and counted under a fluorescence microscope (Nikon, Tokyo, Japan).
The embryos were removed of zona pellucida with 0.5% acidic Tyrode solution, and permeabilized by 1% Triton-X 100/PBS. After permeabilization, embryos were incubated with TUNEL solution from the In Situ Cell Death Detection Kit (Roche; Mannheim, Germany) in the dark at 37 °C for 1 h. Then, embryos were washed three times with PBS/PVA. DNA were stained with DAPI for 15 min. All samples were observed under a fluorescence microscope (Nikon, Tokyo, Japan) after mounting. Each experiment was biologically replicated at least 5 times. The same exposure times and microscope settings were used for all captured images.
Reactive oxygen species (ROS) generation detection
The ROS level of oocytes were detected by a Reactive Oxygen Species Assay Kit (Beyotime Institute, Shanghai, China) following the manufacturer’s instructions. Briefly, 100 denuded oocytes of each group were incubated with dichlorofluorescein diacetate (DCFH-DA) probe (10 µmol/L) in the dark at 38.5 °C for 1 h followed by washing three times with PBS, the oocytes were transferred and mounted on a glass slide, and then were observed with fluorescence microscope (Nikon, Tokyo, Japan) with the same exposure times and microscope settings.
Library preparation and single-cell RNA-seq (scRNA-seq)
The SMART-Seq2 technology was used to detect the global gene expression level in different groups. Briefly, the zona pellucida-free embryos (more than 20 blastomeres) were washed with PBS/PVA, and then were transferred into 0.2 mL tube with 4 µL lysis buffer (including 0.2 µL diluted ERCC spike-in) (1:1000). After SMART amplification, Ampure XP beads were used to purify the cDNA, and Qubit 3.0 Flurometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the concentration of library, Agilent 2100 Bioanalyzer (Agilent Technologies) was used to detect the insert size and quality of amplified library. All libraries (PACL_4cell, PA_4cell, PACL_Bla and PA_Bla) were sequenced by Annoroad Biotechnology (Beijing, China) on an Illumina platform, and generated 150 bp paired-end reads. The clean reads were mapped to Sus_scrofa. Sscrofa11.1 and quantified gene expression levels as TPM (Transcripts Per Million).
Identifying differentially expressed genes (DEGs) and lncRNAs (DElncRNAs)
DESeq2 was used for DEGs and DELncRNAs analysis of RNA-seq with a threshold of false discovery rate (FDR) < 0.05 and |log2FC| >1 between two groups.
Co-expression and Function analysis
The DEGs, target genes of DElncRNAs were performed function enrichment analysis, including GO and KEGG signaling pathway analysis using KOBAS 3.0 software. The lncRNAs-gene interaction networks were visualized with Cytoscape 3.4.0 (http://www.cytoscape.org/).
Predication of m6A sites in mRNA
The m6A sites of genes were predicated with the online software SRAMP (http://www.cuilab.cn/sramp) with default parameters. The very high/high/moderate confidence m6A sites were considered as the predicted m6A sites.
RNA isolation and real-time quantitative PCR (qRT-PCR)
Total RNA was isolated from porcine oocyte or embryos with RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. cDNA was synthesis by Transcript All-in-one First-Strand cDNA Kit (TransGen, Beijing, China), followed by qRT-PCR on a StepOnePlus™ Real-time PCR systems instrument (Applied Biosystems). The qPCR primers were designed by Primer3plus (http://primer3.sourceforge.net/webif.php) and synthesized by Comate biotech Co., Ltd. (Changchun, Jilin, China) (Table S1). In the present study, GAPDH was used as the internal control gene. The 2−ΔΔCt method was used for the calculation of gene relative abundance.
All experiments were performed at least three independent replicates. GraphPad Prism8 statistical software (Graphpad Software, San Diego, CA, USA) was used for data analysis. Graphics were drawn using GraphPad Prism8 and R software (4.0). The results were presented as Mean ± SEM, and analyzed by t-test, one-way ANOVA test and multiple t test. p < 0.05 was considered statistically significant, p < 0.01 was considered extremely significant.