Oocyte collection and in vitro maturation (IVM)
Bovine oocyte collection and IVM were carried out as described previously [22]. The cumulus-oocyte complexes (COCs) were removed from 2-8 mm follicles (obtained from a slaughterhouse) under a stereo microscope (Nikon Ti-E, Japan), and washed three times with phosphate buffered saline (PBS) with 5% fetal bovine serum (FBS). COCs with even cytoplasm and at least a three-layer cumulus were selected for IVM. The culture medium for IVM was TCM-199 bicarbonate buffer with 10% FBS, 1 μg/mL 17ß-estradiol, and 0.075 IU/mL human menopausal gonadotropin (TCM-199, Gibco, BRL, Grand Island, NY, USA). Groups of 300 COCs were cultured in 4 mL medium in humidified air with 7% O2, 5% CO2 at 38.5°C for about 20 h. The cumulus cells outside the zona pellucida of the oocytes were removed with 0.1% hyaluronidase, and oocytes exhibiting the first polar body (Pb) were selected for subsequent experiments.
In vitro fertilization (IVF)
IVF were carried out as previously described [23]. Briefly, frozen semen (Yang Ling Ke Yuan Co. LTD, China) was thawed at 37°C, placed in a 15 mL tube with 5 mL Brackett and Oliphant (BO) medium supplemented with 20 μg/mL heparin and 6 mg/mL BSA, and incubated for 30 min at 38.5°C under 7% O2, 5% CO2. The treated semen supernatant was transferred to a 1.5 mL tube, centrifuged at 1200 g for 5 min and the sperm pellets recovered. The sperms were diluted to 2×106 sperm/mL, and 40 μl aliquots were added to 20-25 matured COCs in a volume of 460 μl BO-IVF medium (IVF Bioscience, Falmouth, United Kingdom). After IVF for 20 h, cumulus cells and excess sperm were removed from the oocytes by incubation in PBS containing 0.1% bovine testicular hyaluronidase. The oocytes with a second Pb were selected for further culture.
Somatic cell nuclear transfer (SCNT)
The procedures for SCNT were carried out as previously described [24]. Briefly, after IVM, cumulus cells were separated from COCs by incubation with 0.1% bovine testicular hyaluronidase in PBS. Metaphase II (MII) oocytes with first Pb were selected and stained with 10 μg/mL Hoechst 33342 for 10 min prior to micromanipulation. Enucleation was performed using a 20 μm glass pipette by aspirating the first Pb and a small amount of the surrounding cytoplasm in a 100 μL micro-drop of PBS supplemented with 10% FBS and 7.5 μg/mL cytochalasin B (CB). The extracted cytoplasm was examined under ultraviolet illumination in another micro-drop to confirm successful enucleation. The nuclear donor cells were ear fibroblasts from fetal Holstein cows that were serum-starved, and nuclei were injected into the perivitelline space of successfully enucleated oocytes. The oocyte-cell couplet was formed by electrofusion. Successfully reconstructed embryos were incubated in mSOFaa containing 5 μg/mL cytochalasin B for 2 h, activated with 5 μM ionomycin for 4 min, then with 1.9 mM dimethynopyridine for 4 h in mSOF. After activation, the embryos were cultured in 50 μL drops of mSOFaa medium supplemented with 8 mg/mL BSA under a humidified atmosphere of 7% O2, 5% CO2 at 38.5°C. The cleavage rate was determined at 48 h after culture, and blastocyst formation was counted on day 7 of culture.
Microinjection
The concentration of double-stranded miR-183 mimic / miR-183 mimic control for microinjection (Transgen, Beijing, China) was adjusted to 2 ng/μL as described previously [25]. Double-stranded RNA is necessary to increase stability during the experimental procedure and to facilitate the formation of the miRISC in cells. EZR-siRNA, EZR-siRNA control, Slc9a3r1-siRNA and Slc9a3r1-siRNA control were produced by GenePharma (Shanghai, China). About 2 μL of injection solution was loaded into a microinjection needle (Eppendorf, Hamburg, Germany). The embryos for injection were transferred into PVA micro-droplets and covered with mineral oil. Changes in the cytoplasm were monitored during injection to ensure that the pulse pressure and injection volume of the experimental group and the control group were as consistent as possible. Injected embryos were then transferred into pre-balanced mSOF medium and cultured at 38.5°C in an atmosphere of 7% O2, 5% CO2.
Dual luciferase reporter gene assay
For the testing of target genes using TargetScan, PicTar, miRanda websites and the relevant literature (Supplementary data 7), EZR (ezrin gene), Pdcd6 and Ppp2ca were selected as candidate targets of miR-183 after KEGG pathway analysis (Supp. Data 8). For the target gene test, the 3'-UTR regions of the target genes were amplified using fetal bovine liver tissue as cDNA template. The primer sequences are given in Table 1 and Table 2. Each 50 μL PCR reaction contained DNA polymerase (TransStart FastPfu, Beijing, China), 1 μL; 5×buffer (TransStart FastPfu, Beijing, China), 10 μL; forward primer (10 mM), 1 μL; reverse primer (10 mM), 1 μL; dNTPs (10 mM), 4 μL; template, 1 μg; dd H2O, 35 μL. The PCR reaction procedure was as follows: 94°C for 3 min; (94°C for 30 s, 60°C for 30 s, 72°C for 30 s) 40 cycles; 72°C for 10 min, 16°C for 10 min. The sequence and pSicheck-2 carrier involved a dual enzyme reaction with restriction endonucleases NotI and XhoI, followed by ligation. Afterwards, the target gene reporter carriers were constructed: psi-EZR, psi-Pdcd6, psi-Nck2, psi-Ppp2a, psi-Crk, psi-Teme150a, psi-Srek1ipi, psi-Map3k4, psi-Slc35a1. With the target gene reporter carriers as templates, the target gene psi-mutants, psi-mut-EZR, psi-mut-Pdcd6 and psi-mut-Ppp2a, were successfully constructed.
The dual luciferase reporter assay was performed using a TransDetect double-luciferase reporter assay kit (TransGen Biotech, China) following the manufacturer’s procedure. Briefly, Renilla luciferase substrate and firefly luciferase substrate were thawed at 16°C. Cells in 24-well plates were washed three times with pre-cooled saline, mixed with 100 μL of lysis buffer and allowed to lyse for 15 min at room temperature. Lysates were centrifuged at 12,000 g for 10 min at 4°C, supernatants were mixed with firefly luciferase substrate, and 20 μL aliquots were added to each well of a 96-well plate. Fluorescence values of the firefly luciferase reporter gene were measured with a chemiluminescence apparatus, with three well repeats for each sample. Afterwards, the Renilla luciferase substrate was added, and the fluorescence values of the Renilla luciferase reporter gene were detected with the chemiluminescence instrument.
Immunofluorescence staining of embryos
The embryos were washed three times with PBS containing 0.2% PVA (PBS/PVA) and fixed overnight in 4% paraformaldehyde at 4°C. After washing with PBS/PVA three times, five mins each time, they were transferred into PBS/PVA containing 0.1% Triton X-100 for permeabilization, 30 min at 25°C. After washing, the embryos were kept in blocking buffer (Beyotime P0102, Shanghai, China) for three hours at room temperature. After washing, the embryos were incubated with the primary antibody (Abcam, Cambridge, UK) overnight at 4°C. After washing, they were incubated with secondary antibody (Beyotime, Shanghai, China) at room temperature in the dark for 2 h. The DNA was stained with 4, 6-diamidino-2-phenylindole (DAPI), (Beyotime C1005, Shanghai, China) for 3-5min. Finally, the embryos were placed on glass slides and examined using a Nikon Eclipse Ti-S microscope equipped with a 198 Nikon DS-Ri1 digital camera (Nikon, Tokyo, Japan).
The Dead End TM colorimetric TUNEL system (Promega, Madison, WI, U.S.) was used to detect blastocyst apoptosis. The blastocysts were fixed overnight in 4% paraformaldehyde at 4°C, permeabilized in 0.1% Triton X-100 for 30 min at 25°C, transferred into E buffer micro-droplets at room temperature for 5 min, and incubated with FITC-conjugated dUTP and terminal deoxynucleotidyl transferase at 37°C for 1 h in the dark. 2×SSC (SSC: 0.15 mol/L sodium chloride and 0.015 mol/L sodium citrate) was added to stop the reaction and samples were allowed to stand for 15 min. Samples were stained with DAPI at room temperature in the dark for 8 min. Staining was observed under a fluorescence microscope (Nikon, Tokyo, Japan).
Reverse transcription and qRT-PCR
Total RNA was isolated from about 10 SCNT embryos for each experiment. RNA reverse transcription was performed using the miScript II RT kit according to the manufacturer’s directions (Qiagen, Germany). Briefly, we mixed 2 μL of 10×miScript Nucleics mix, 4 μL 5×miScript Hispec buffer, 10 μL purified RNA, 2 μL miScript reverse transcriptase mix and 2 μL RNase-free water for each 20 μL reaction. Thermocycling conditions were as follows: 37°C for 60 min and 95°C for 5 min. Primer sequences are listed in Table 3. The reverse transcription process was performed according to the instructions for the TransScript first-strand cDNA synthesis super-mix (Transgen, Beijing, China). The procedure was as follows: 25°C, 10 min; 42°C, 30 min; 85°C, 5 min. Quantitative RT-PCR was performed according to the procedure for the SYBR premix Ex Taq (Perfect Real-Time), and the primers were synthesized at Xi 'an Qing Ke. The PCR procedure is as follows: 95°C, 30 s; (95°C 5s, 60°C 30 s, 95°C 15 s) 35 cycles; 60°C, 5 min. The 2-△△Ct method was used to calculate the relative expression level of the target mRNAs and statistical comparisons were conducted by one-way ANOVA.
Protein identification by western immunoblot
Aliquots of protein extracts were run at a constant voltage of 80V on an SDS-PAGE gel until the samples entered the separating gel, at which time it was switched to a constant voltage of 120 V. The gel containing the target proteins and size markers was blotted to PVDF film for 120 min at a constant current of 250 mA and 4°C with blocking buffer, overnight at 4°C. The first antibody was diluted and the blot was incubated at 4°C for 12 h. After washing twice with 1×TBST, the second antibody was diluted and incubated with the blot at room temperature for 2 h in the dark.
Co-IP
Primer 5 software was used to analyze and synthesize the upstream and downstream primer sequences of pCMV-HA-Slc9a3r1 and pCMV-EGFP-EZR vectors (Supp. Data 9). PCR amplification was performed as described in Section 2.5. The reaction conditions were as follows 95°C for 5 min, (95°C for 30 s, 56°C for 30 s, 72°C for 2 min) 30 cycles, 72°C for 10 min. The amplified DNAs were run on agarose gels, the bands were cut out and the DNAs were extracted (OXOID, UK). Restriction endonucleases, NheI (GCTAGC) and AgeI (ACCGGT) were used to perform a double digest of the PCR-amplified product and the pCMV-EGFP skeleton vector, respectively. At the same time, BglII (AGATCT) and NotI (GCGGCCGC) were used for double digestion of the PCR amplified product and the pCMV-HA skeleton carrier, respectively. The reaction conditions were as follows: 100×BSA, 0.5 μL; 10×NEB buffer, 5 μL; DNA, 5 µg; enzyme A, 0.5 μL; enzyme B, 0.5 μL; dd water to 50 μL, incubate at 37°C for 8 h. The ligation was performed with a DNA ligation kit: dd H2O, 4 μL; linearized vector, 1 μl (45-150 ng); cDNA, 1 μl (45-100 ng), Quick-Clone mix, 4 μL. The reconstructed carrier was used for transfection, and identified by enzyme digestion (KpnI-XhoI was used for double digestion of pCMV-EGFP-EZR, and BglII-NotI was used for double digestion of pCMV-HA-Slc9a3r1) (Promega, USA) and PCR, the forward primer and reverse primer sequences of pCMV-HA-Slc9a3r1 and pCMV-EGFP-EZR plasmid carrier were designed, and the primer sequences are given in Supp. Data 10. The PCR reaction procedure was as follows: 10×TransStart Taq buffer, 2 μL; Trans Fast Taq DNA polymerase, 0.2 μL; P1 (universal primer), 0.4 μL; P2 (universal primer), 0.4 μL; dNTPs (10 mM), 0.4 μL; take a small amount of bacterial colony on a micropipette tip as template; dd H2O, 16.6 μL. 95°C for 5 min, (95°C for 30 s, 56°C for 30 s, 72°C for 2 min) 30 cycles, 72°C for 10 min. After successful sequencing, endotoxin-free plasmid extraction was carried out (Promega, USA). The three combinations of ezrin-GFP & HA-empty, GFP-empty & HA-SLC9A3R1 and ezrin-GFP & HA-SLC9A3R1 were transfected into 293T cells using Lipofectamine 2000 (Invitrogen, USA). Total protein was extracted from each group by incubating in a boiling water bath for 5 min, an ice bath for 2 min, and centrifugation for 2 min at 20,000 g, followed by SDS-PAGE electrophoresis. Ezrin-GFP & HA-SLC9A3R1 protein were extracted and mixed with 10 μL anti-HA-tag magnetic beads, and the other group was mixed with 10 μL anti-GFP-tag magnetic beads. After reversing and mixing to balance the beads, they were centrifuged at 5,000 g for 15-30 s at 4°C and supernatants were removed. The supernatants were mixed with 10 μL anti-HA-tag magnetic beads (another group of protein supernatants were mixed with 10 μL anti-GFP-tag magnetic beads), mixed evenly, and incubated at 4°C for 3 h. Centrifugation was performed at 5,000 g, 4°C for 30 s and supernatants were removed. Pellets were washed four times with 1 mL aliquots of wash buffer, 5 min each time. The pellets containing immunoprecipitated proteins were resuspended in 35 μL SDS loading buffer, placed in a boiling water bath for 5 min, then on ice for 2 min, followed by centrifugation for 2 min at 20,000, and running on an SDS-PAGE gel.
Preparation of embryo samples for scanning electron microscopy
Embryo samples from each group were collected and fixed in 2.5% glutaraldehyde at 4°C overnight. Before dehydration, the embryo samples were washed four times, 10 min each, with 1M PBS, pH 7.2. Fixed/washed embryo samples were successively placed in 30%, 50%, 70%, 80% and 90% ethanol for 15 min each time, then dehydrated in 100% ethanol three times, 30 mins each time. After critical-point drying and metal spraying, the microstructure of the embryo microvilli was observed under a scanning electron microscope (Leica TCS SP8, Germany).
Statistical analysis
Data were averaged from at least three independent experiments. Three technical replicates were applied for RT-qPCR to detect the expression in the different groups. The comparative threshold cycle method (2-△△Ct) was used to calculate the relative expression of miRNAs. Statistical comparisons were conducted by one-way ANOVA. Immunofluorescence intensity was determined with Image J software (National Institutes of Health, Bethesda, MD, USA). Mean gray value (Mean) = integrated density (IntDen) / area. Statistical analyses were conducted using the SPSS software package (SPSS Inc, Chicago, IL, USA). Data were expressed as means ± standard errors of the mean (SEM), and p < 0.05 was considered statistically significant.