2.1 Follicle Collection
Ovaries were obtained from mature sows at a local slaughterhouse. Individual antral follicles, approximately 3 to 5 mm in diameter, were dissected from the ovaries after quickly washing twice with 75% ethanol and physiologic saline using small scissors and fine forceps and then classified into healthy follicles (HFs) and atretic follicles (AFs) according to follicle shape, GC density, and hormone levels (Zhang et al., 2018a).
2.2 Cell Culture and Transfection
Primary GCs were obtained from HFs by syringing with a 20-gauge needle and cultured as previously described (Liu et al., 2018). HEK293 cells were cultured as previously described (Liu et al., 2018). VEGFA siRNA, Smad4 siRNA, miR-361-5p mimic, and miR-361-5p inhibitor were synthesized by GenePharma (Shanghai, China) (Supplementary Table S1). After 12 h of culture, the porcine GCs were transfected with the appropriate plasmids or oligos using Lipofectamine 2000 and Opti-MEM (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.
2.3 Immunohistochemical assay
To examine the expression and location of VEGFA in healthy and atretic follicles, immunohistochemical staining was performed according to our previous description(Gao et al., 2020). Rabbit polyclonal VEGFA (diluted 1: 200 in PBS containing1% (W/V) bovine serum albumin, ab9570, Abcam, Cambridge, MA, USA) and secondary antibody (G1210-2-A, Servicebio, Wuhan, China) was applied. The specific protein immunoreactivity was visualized with 0.05% 3,30-diaminobenzidine (DAB, G1211, Servicebio, Wuhan, China) for 15 min, and the slides were counterstained with hematoxylin (G1004, Servicebio, Wuhan, China). The images were captured under the microscope (Nikon Eclipse E200, Tokyo, Japan).
2.4 RNA extraction and qRT-PCR
Total RNA was extracted from the follicles and GCs using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Then the extracted total RNA was reverse transcribed to cDNA by Super M-MLV RTase Synthesis Kit and qRT-PCR was performed using SYBR Premix Ex Taq (Takara, Dalian, China) on the ABI StepOne system (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's instructions. GAPDH was used as an internal control. For miRNA detection,frst-strand cDNA synthesis and qRT-PCR were performed using miRNA two-step qRT-PCR SuperMix (TransGen, Beijing, China). U6 was used as internal control. For each gene, controls for each primer set containing no cDNA were included on each plate, and the reaction was repeated three times for every sample. The primers for qRT-PCR are listed in Supplementary Table S2.
2.5 fluorescent in situ hybridization (FISH)
The FAM-labeled probe (5'-GTACCCCTGGAGATTCTGATAA-3 ') was specifically synthesized for miR-361-5p, and DAPI was used to stain the cell nuclei. The GCs were cultured on coverslips, fixed in 4% paraformaldehyde (containing DEPC) for 20 min, washed with shaking in PBS (PH7.4) for three times, and proteinase K (20ug/ml) was finally added for 5 min for digestion. Then all the procedures were conducted according to the manufactory’s instruction (Sevicebio, Wuhan, China). Finally, the images were acquired on a Nikon upright fluorescence microscope (Nikon DS-U3, Japan). Each experiment was performed three times.
2.6 Protein extraction and western immunoblotting analysis
GCs were washed with cold PBS and lysed with RIPA buffer containing 1% phosphatase inhibitor (v/v) (Beyotime, Shanghai, China) and proteinase inhibitor (Sigma, St. Louis, MO, USA). The protein concentration was determined by the BCA Protein Assay Kit (Beyotime, Shanghai, China) and diluted to the same concentration using the 5 × Protein Loading Dye (Sangon, Shanghai, China). Total protein extracts were separated using SDS-PAGE on 12% gels. The proteins were then transferred onto the PVDF membranes (Millipore, Billerica, MA, USA), and the membranes were blocked with 5% non-fat milk for 2 h. After washing with Tris-buffered Saline with Tween (TBST) for 15 s, the membranes were incubated with anti-VEGFA (diluted 1: 5000, ab9570, Abcam, Cambridge, MA, USA), anti-Tubulin (diluted 1:1000, 10094-1-AP ProteinTech, Nanjing, China), anti-caspase3 (diluted 1:1000, 19677-1-AP, ProteinTech, Nanjing, China) overnight at 4 °C. Then incubated with a secondary peroxidase-conjugated antibody (diluted 1:2000, Cell Signaling Technology, Beverly, MA, USA) for 1 h at room temperature. Chemiluminescence was detected by WesternBright™ ECL (Advansta, Menlo Park, CA USA) and analyzed using Image J software. Each experiment was performed three times.
2.7 Plasmid construction
The 3′-UTR fragments of VEGFA containing putative target sites of miR-361-5p and the promoter fragments of the miR-361-5p coding gene (MIR361) containing putative SMAD4 binding sites were amplified from porcine genomic DNA and verified by sequencing. The VEGFA 3′-UTR fragment was digestion with Nhel and Xbal (Thermo, Waltham, MA, USA), and then cloned into pmirGLO Dualluciferase miRNA Target Expression Vector (Promega Corporation, Madison, WI, USA). The MIR361 promoter fragment was digestion with NheI and SacI and then cloned into pGL-3 reporter vector (Promega, USA). The mutant plasmids of the miR-361-5p putative binding site were generated by ClonExpress Entry One Step Cloning Kit (Vazyme, Nanjing, China) according to the manufacturer’s protocol. The successful mutations were identified by sequencing technology. The overexpression plasmid pcDNA3.1-SMAD4 plasmid was generated previously by our group (Du et al., 2018). Primers used here are detailed in Supplementary Table S3.
2.8 Luciferase Reporter Assays
After a transfection period of 24 h, the cells and lysates were collected. A Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) was used to quantify luciferase activities following the manufacturer’s instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. Each experiment was performed six times.
2.9 Apoptosis assay
GCs apoptosis was measured by annexin V-FITC/PI staining assay (Vazyme, Nanjing, China) according to the manufacturer’s protocol. A cell-counting machine (Becton Dickinson, USA) was used for the detection of apoptotic cells based on the principle of fluorescence-activated cell sorting (FACS). The data were analyzed using FlowJo v7.6 software (Stanford University, Stanford, CA, USA).
2.10 Statistical analysis
All data are presented as means ± S.E.M. Prism 5 software (GraphPad Software) was used to perform statistical analysis. Two-tailed Student’s t-test was used to evaluate the significance when two groups were compared. When three or more groups were compared, a one-way analysis of variance test was performed, and Turkey’s test to determine significance between groups. P-value of < 0.05 and 0.01 was considered significant and extremely significant differences, respectively.