Chemicals
Unless otherwise mentioned, all chemicals used in the experiments were procured from Sigma-Aldrich Company (MO, USA).
Preparation of IVM media
TCM-199 medium was supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Isle, NY), 10% (v/v) porcine follicular fluid collected from antral follicles (diameter ≥ 8 mm) of slaughter house-derived ovaries, 10 ng/mL epidermal growth factor, 0.6 mM cysteine, 10 IU/mL equine chorionic gonadotropin, and 10 IU/mL human chorionic gonadotropin.
Preparation of IVTMOs
Porcine ovaries were obtained from a local slaughterhouse and transported to the laboratory in 0.9% saline (w/v) within three hours. The NaCl solution was supplemented with penicillin-G (100 IU/mL) and streptomycin sulfate (100 mg/L) at 30℃-35℃. Follicular fluid with the oocytes was aspirated from the antral follicles (3–6 mm in diameter) and collected into a 50 mL centrifuge tube using an 18-gauge needle connected to a 10mL disposable syringe. Cumulus-oocyte complexes (COCs) were recovered under a stereomicroscope. The samples with at least three layers of compact cumulus cells and a homogenous cytoplasm were selected for IVM. The selected COCs were washed thrice with HEPES-buffered Tyrode’s medium containing 0.05% (w/v) polyvinyl alcohol. Approximately 50–60 oocytes were transferred to each well of a 4-well Nunc dish into 500µL IVM media supplemented with different concentrations of SDF-1, and cultured at 38.5℃ with 5% CO2 for 44 hours in a humidified atmosphere. The COCs were treated with DPBS containing 1 mg/mL hyaluronidase to remove the surrounding cumulus cells. The oocytes were observed under a stereoscope, and those with the first polar body in the perivitelline space were considered as mature. The mature oocytes with intact cell membranes and clear perivitelline space were selected for SCNT, IVF and PA.
Preparation of donor cells
Fibroblast cells were obtained from a Duroc boar by ear biopsy. After a quick rinse in 75% ethanol and DPBS supplemented with penicillin (100 IU/mL) and streptomycin (100µg/mL), the ear tissues were minced into 1 to 2 mm pieces in DMEM supplemented with 10% FBS. Fragments of ear tissue were seeded into 100 mm cell culture dishes in 6 mL FBS-supplemented DMEM (Invitrogen, Grand Island, NY, USA), which were then placed in a humidified incubator at 38.5°C with 5% CO2. The culture medium was changed every other day. The fibroblasts were harvested with 0.25% trypsin-EDTA (Invitrogen, Grand Island, NY, USA) and passaged at 1:3 split ratios (passage number 1, P1). The fibroblast cells from passages 3–5 were frozen in liquid nitrogen (freezing medium − 50% FBS, 40% DMEM and 10% DMSO). Before SCNT, the cells were thawed and cultured as described in FBS-supplemented DMEM for 3–4 days until they reached 80%-90% confluence. Adherent cells from passages 6–10 were harvested with 0.05% trypsin for 1 minute and used for SCNT.
Production of embryo
Somatic cell nuclear transfer (SCNT): The matured oocytes and a small number of donor cells were suspended in T2 medium (TCM-199 plus 2% FBS) containing 7.5µg/mL cytochalasin B (CB). The oocytes were enucleated by microinjection using a 17µm diameter needle (Lingen Precision Medical Products Co., Ltd., Shanghai, China). The microinjection needle was inserted into the oocyte to aspirate the first polar body along with approximately 15% of the adjacent cytoplasm that contains the genomic material. Donor cells with a round and slightly burr-like shape were aspirated into the injection needle, and one donor cell was injected into the perivitelline space of each enucleated oocyte. The reconstructed oocytes were washed three times in PZM-3 and then electrically fused using two direct current pulses of 150 V/mm for 50 minutes in 0.28 mol/L mannitol supplemented with 0.1 mM MgSO4 and 0.01% PVA. The fused oocytes were cultured in PZM-3 medium for 1 hours before electro-activation in a medium supplemented with 5 mg/mL CB. The cells were then incubated at 38.5℃ under 5% CO2 and saturated humidity for 4 hours. The reconstructed oocytes were activated by two direct current pulses of 100 V/mm for 20 minutes in 0.28 mol/l mannitol supplemented with 0.1 mM MgSO4 and 0.05 mM CaCl2.
Parthenogenesis: The matured oocytes were washed three times in PZM-3 and then electrically activated using two direct current pulses of 150 V/mm for 50 minutes in 0.28 mol/L mannitol supplemented with 0.1 mM MgSO4 and 0.01% PVA. The activated oocytes were then cultured in PZM-3 medium supplemented with 5 mg/mL CB for 4h at 38.5°C under 5% CO2 and saturated humidity.
In vitro fertilization (IVF): The matured oocytes were transferred to mTBM medium (11.0 mM glucose, 5.0 mM sodium pyruvate, 113.1 mM NaCl, 7.5 mM CaCl2·2H2O, 3.0 mM KCl, 20.0 mM Tris) supplemented with 2 mg/mL BSA and 2.5 mM caffeine. The fresh semen (Guangdong Wens Breeding Swine Technology Co., Ltd., Guangdong, China) was washed three times by centrifugation with DPBS containing 0.1% BSA at 1500 rpm for 5 minutes. The spermatozoa were resuspended and capacitated with mTBM in the CO2 incubator for 30 minutes. The capacitated sperm were added to the well containing matured oocytes with a final concentration of 1 × 105 sperm/mL for 6 hours at 38.5°C under 5% CO2 and saturated humidity.
The embryos generated by SCNT, PA and IVF were transferred to fresh PZM-3 for extended culture, and the cleavage and blastocyst rates were assessed two and six days after embryo production. The total blastocyst cell count was determined by Hoechst 33342 staining under an epifluorescence microscope (Ti2; Nikon).
RNA sequencing and bioinformatics analysis
The matured oocytes were washed thrice with DPBS at 37.5℃. Fifty randomly selected oocytes were pooled and transferred into a 200µL PCR tube containing 10µL RNA lysis buffer, and then stored at -80℃. Oocyte RNA extraction, library construction and transcriptome sequencing were conducted by OE Biotech Co., Ltd. (Shanghai, China). Each group had three biological replicates, and six samples were sequenced. Filtered clean reads were mapped to the Sus scrofa 11.1 reference genome. The differentially expressed genes (DEGs) between the control (0 ng/mL) and 20 ng/mL SDF1-supplemented (20 ng/mL) groups were identified using R package DEseq2[46]. Gene Ontology (GO) analysis of the DEGs was performed by ClueGO and CluePedia plug-in of Cytoscape. The R package clusterProfiler was used for Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis[47].
Real-time quantitative polymerase chain reaction (qPCR) analysis
Total RNA was extracted from the matured oocytes using the complete RNA Kit (Omega, Atlanta, USA), and quantified using NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). Only intact RNA samples that did not show any signs of degradation were stored at − 80°C till subsequent analysis. The cDNA was synthesized from 1µg total RNA using the PrimeScript RT Reagent Kit (TaKaRa, Otsu, Japan), and qRT-PCR was performed using the PowerUp™ SYBR Green Mix (Applied Biosystems, MA, USA) according to the manufacturers’ protocols. The primer sequences of all transcripts are listed in Table S1. The expression of each target gene was quantified relative to that of the internal control gene (RN18S) using the equation R = 2 −[△Ct sample−△Ct control].
Measurement of ROS levels in oocytes
The ROS levels in the oocytes were measured using the ROS Assay Kit (Yesen, Shanghai, China) as per the manufacturer’s instructions. The oocytes were incubated with 10µM 2′,7′-dichlorodihydrofluorescein diacetate (D2DCFDA) in M199 medium at 38.5℃ for 15 minutes in the dark. After washing twice with M199 medium at room temperature, the oocytes were transferred into 30µL DPBS droplets, and the fluorescence was observed using an epifluorescence microscope (Ti2; Nikon). The fluorescence intensity was analyzed by ImageJ software (National Institutes of Health, USA).
Detection of mitochondrial membrane potential (ΔΨm) in oocytes
The ΔΨm in the oocytes was measured using JC-1 mitochondrial membrane potential assay kit (Abcam, Cambridge, Britain). The oocytes were incubated with 10µM JC-1 in medium at 37℃ for 40 minutes in the dark. The samples were washed thrice with DPBS, and the fluorescence intensity was detected with an epifluorescence microscope using ImageJ software. The ΔΨm was calculated as the ratio of red to green fluorescence.
Immunofluorescent staining of IVTMOs
The matured oocytes were fixed with 4% paraformaldehyde (Beyotime, Shanghai, China) for 15 minutes and permeabilized in 0.5% Triton X100 (Beyotime, Shanghai, China) for 20 minutes. After blocking with QuickBlock™ blocking buffer (Beyotime, Shanghai, China) for 30 minutes, the oocytes were washed thrice with DPBS and incubated overnight with primary antibodies targeting TET1 (Bioss, Beijing, China) and YY1 (Proteintech, Chicago, USA) diluted 1:100 at 4°C. The oocytes were washed thrice with DPBS, and then incubated with Alexa Fluor 488 goat anti-rabbit IgG or Cy3–conjugated affiniPure goat anti-mouse IgG diluted 1:200.
Statistical analysis
Statistical analyses was performed using SPSS 21.0 (SPSS, Inc., Chicago, IL, USA). The groups were compared by ANOVA or Student's t-test as appropriate, and P < 0.05 was considered statistically significant. All data are presented as the mean ± standard error (SEM).