Isolation, culture and identification of primary endometrial cells of dairy cows
This study was performed in accordance with the Guidelines of the Animal Ethic Committee of Beijing University of Agriculture under the permit No. SYXK (JING) 2015-0004. The uterus of healthy cows was taken from the slaughterhouse, stored in DPBS containing 4% penicillin and transported to the laboratory at 37oC. Then the tissue was cut to select the uterus-free carcass tissue, which was then washed with DPBS for 2-3 times and cut into small pieces. The endometrial epithelial cells (EECs) were isolated with the tissue migration method, and passed through 3-4 passages successively. Fibroblasts were removed with 0.25% pancreatin and differentially digested at 37oC. The purity of EECs was identified by detecting the expression of keratin 7 protein with the immunofluorescence method.
In vitro maturation of oocytes
The bovine ovaries were taken from the slaughterhouse, placed in the cleaning solution of penicillin and streptomycin, transported back to the laboratory within 2~4 h, and washed repeatedly in the laboratory. Then a 20 mL syringe was used to aspirate the follicular fluid from a follicle with a diameter of 3 to 8 mm and pour it into a 60 mm Petri dish. A high-quality cumulus-oocyte complex (The cumulus has three layers or more and is densely wrapped without diffusion) was selected under the microscope and washed for three times in the HEPES droplets, before it was put in the pre-balanced IVM mature droplets, washed for three times, and transferred to and incubated in an incubator with 5% CO2 at 38.5oC for 22 h.
The in vitro matured oocytes, 1 mL of HEPES egg wash, and 200 μL of 0.1% hyaluronidase were all added to a 10 mL centrifuge tube which then was shaken with a shaker for 4 min. The granulosa cells around the mature oocytes were removed. Next, the oocytes that have been expelled from the first polar body were picked out and moved into the ION droplets to protect them from light for 5 min. After activation, it was transferred into the 6-DMAP droplets, washed twice, then cultured in the 6-DMAP droplets for 4 h, and finally transferred into the embryo culture medium which was cultured in a saturated humidity environment with 5% CO2 at 38.5oC. The embryo culture medium (mCR) containing 10% FBS was changed in half every 48 h, and the cleavage rate was counted after 48 h after the parthenogenetic activation operation. The day when the parthenogenetic activation started was set to the zeroth day, and the blastocyst rate was counted till the 7th day.
In vitro fertilization
After in vitro maturation, the oocytes were washed twice with the HEPES egg wash, and then moved into the pre-balanced fertilization droplets. Each drop of 10~15 oocytes was combined with 1~2 pieces of frozen semen (Beijing Dairy Cow Center: Limousin (No. 11108016)), shaken in the air for 10 s, and then thawed in a 37oC water bath. The motility and density of sperms were checked. The thawed semen was slowly added to the sperm wash, and centrifuged at 1,800 rpm for 8 min twice. After the supernatant was discarded, the sperm concentration was adjusted to 2~4×106/mL for in vitro fertilization. The treated frozen semen was injected into the fertilized droplets containing 15 μL mature oocytes per drop, which was placed in a saturated humidity incubator with 5% CO2 at 38.5oC. After the incubation of a total of 18 h, the early embryos after fertilization were separated from the fertilization fluid, and placed and shaken on a shaker. Next, the HEPES egg washing solution was added to the highest level of the shaker, and centrifuged at 1,000 rpm for 1 min. The embryos sucked out of the tube were washed twice with HEPES and cultured in a saturated humidity environment with 5% CO2 at 38.5°C. The late embryo culture medium containing FBS was changed once every 48 h. The cleavage rates of in vitro fertilized embryos were counted after 48 h after fertilization. The day when fertilization started was set to the zeroth day, and the blastocyst rate was counted up to the 8th day.
Somatic cell nuclear transfer
The fresh bovine ears were collected from the slaughterhouse, and the tissue block migration method was used to obtain the bovine ear fibroblasts, and the microinjection method to drive the donor bovine ear fibroblasts into the enucleated oocytes to prepare the reconstructed embryos, which were then placed in the electrofusion tank, and activated with 250 V/cm and 2DC fusion parameter alternating current for 10 μs. After the electrofusion, the embryos were put into the incubator with ION (ionomycin) in the dark for 5min, and finally transferred to the 100μL 6-DMAP droplets which then were chemically activated in the incubator for 4 h, transferred to M1 for cultivation for 48 h, and finally replaced in half with M2 late culture.
IFNAR-IN-1 hydrochloride (IN-1) concentration screening
Eight-cell IVF embryos were incubated with 10 μM IN-1 to the blastocyst stage, and then with EECs for 48 h. Co-cultured EECs were collected, and total protein from the EECs was subjected to the detection of the expressions of IFNAR1, IFNAR2, Integrin αv, Integrin β3 and WNT7A with the western blot method, respectively. β-actin served as the control gene in the all samples. In addition, the EECs were cultured with 5 μ M IN-1 antagonist and 0.01 ng/mL IFNT for 24 h. Then total mRNA from EECs was subjected to the detection of the expressions of ISG15 and IRF9 with the qRT-PCR method.
IVF embryos with INFAR1 and IFNAR2 knockout embryos obtained
The designed sgRNA1-F, sgRNA2-F, sgRNA3-F, sgRNA4-F, and sgRNA-R (Table S1) were biosynthesized, amplified in vitro, purified by phenol/chloroform, and then combined with the whole genome of EECs after the amplification of sgRNA1, sgRNA2, sgRNA3, and sgRNA4 targets for spCas9 activity in vitro biopsy. RT-PCR was used to detect any target cleavage activity. The effective sgRNA1 and sgRNA4 were microinjected into the embryo after 10 h of sperm-egg combination, and continued to develop to the blastocyst stage. After that, the blastocysts were incubated with EECs for 48 h and collected for T7E1 digestion reaction. RT-PCR was used to detect any genomic target cleavage activity.
Screening of INFAR1 and IFNAR2 gene CRISPR/Cas9 knockout cell lines (IFNAR1-/IFNAR2-), (IFNAR1-/IFNAR2+) and (IFNAR1+/IFNAR2-)
Three pairs of INFAR1 and IFNAR2 gRNA sequences (Table S2) were designed, and then ligated into the PX459 plasmid. Three dual-target plasmids were constructed as I1: sgRNA1-1 + sgRNA2-1; I2: sgRNA1-2 + sgRNA2-2; I3: sgRNA1-3 + sgRNA2-3. Sanger sequencing was performed with U6 primers. The sequencing results confirmed that the two sgRNA fragments in the three plasmids had been ligated to the PX459 plasmid. The INFAR1 and IFNAR2 dual-target recombinant vector was transfected into the EECs with Lipofectamine 3000. After puromycin selection, the picked monoclonal cells were cultured by adding MEM to maintain the culture medium. When the confluence reached 80%-90%, the EECs were collected to verify the expression of INFAR1 and IFNAR2 proteins with Western blot.
INFAR1 and INFAR2 gRNA sequences (Table S3) were designed, and then ligated them into the PX459 plasmid. To construct INFAR1 and INFAR2 target plasmids, we used U6 primers for Sanger sequencing the correct sequencing results of PX459-sgRNA3 (IFNAR1-/IFNAR2+) and PX459-sgRNA4 (IFNAR1+/IFNAR2-) which were then transfected into EEC with Lipofectamine 3000 and into EEC with puromycetes in turn. After the element screening, the picked monoclonal cells were cultured by adding MEM to maintain the culture medium. When the confluence reached 80%-90%, the EECs were collected to verify the expression of INFAR1 and INFAR2 proteins with Western blot.
Co-culture of blastocysts with EECs
The purified and identified primary EECs were sub-cultured, and the bottom of the culture dish was fused into a single layer of cells. After the embryos were parthenogenetically activated, the in vitro fertilized embryos were harvested, the blastocysts (five blastocysts in each group) obtained in vivo and somatic nuclear transfer blastocysts were placed in a single layer of EECs and co-cultured for 24 h. Finally, the embryos and EECs were collected for later use.
RNA extraction and RT-PCR
The samples of blastocyst and EECs were collected separately. Total RNA was extracted with Trizol reagent (Invitrogen) from the fresh tissues according to the manufacturer’s protocol, followed by the removal of genomic DNA with RNase-free DNase (Promega, Madison, WI). After 15 reverse transcriptions, IFNτ, ISG15, WNT7A, IFNAR1, IFNAR2 and β-actin were amplified with PCR. The PCR reactions were conducted in a total volume of 10 µL. The target gene was amplified for 30 cycles of denaturation at 94°C for 30 s, annealed at 57°C for 30 s, and extended at 72°C for 30 s. This was followed by a final 5min extension step at 72°C. The amplified products were analyzed with electrophoresis on 1.5% agarose gels stained with ethidium bromide. The primers used for the analysis were shown in Table S4.
Real-time detection of RT-PCR (qRT-PCR)
Total RNA of each treated cell was extracted with the Trizol (Invitrogen, Inc., Carlsbad, CA, USA) and cDNA was synthesized with the PrimeScriptTM RT Reagent Kit (TaKaRa Bio, Inc., Dalian, China), according to the manufacturers’ protocols, respectively. Real-time PCR was subsequently performed with an ABI 7500 Sequencing Detection System and SYBR Premix Ex Taq™. The GenBank accession numbers and primer sequences of ISG15, IRF9, Integrin αv, Integrin β3, WNT7A and GAPDH are summarized in Table S5. All reactions were performed in at least three independent experiments and the calculated number of copies of target genes was normalized to the number of GAPDH mRNA copies in the same sample.
The blastocysts or EECs were fixed in 4% PFA at 4°C for 24 h. Subsequently, the cells were permeabilized with 0.1% TritonX‐100 for 10 min, blocked with 5% BSA for 30 min, and exposed to anti‐IFNτ, IFNAR1, IFNAR2 and Keratin primary antibody (1:1000 diluted, Abcam, Cambridge, England) at 4°C overnight. After being washed with PBS three times, the blastocysts or cells were incubated with the fluorescent secondary antibody (1:2000 diluted, Beijing Zhongshan Jinqiao Biotechnology Co., LTD, Beijing, China) in the dark at 37°C for 1 h, and then with PI or DAPI at room temperature for 5 min. Finally, they were examined under a laser scanning confocal microscope (TCS SP8 STED; Wetzlar, Hessen, GER).
The proteins were extracted from the frozen blastocysts and EECs with lysis buffer (Nanjing KeyGen Biotech, Nanjing, China). Protein was quantified with the bicinchoninic acid (BCA) protein assay kit (Nanjing KeyGen Biotech). Equal amounts of protein (20 µg) were separated via the 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electro-transferred to 0.22 μm polyvinylidene difluoride membranes (Millipore, Bedford, MA). After the transfer above, the membranes were blocked in the TBST supplemented with 10% skim milk at room temperature for 1 h, and incubated with the relevant primary antibodies at 4°C overnight (Table S6). The next day, the membranes were incubated with the HRP‐labeled secondary antibodies (1:5000 dilutions) at room temperature for 1 h. The immunoreactive bands were visualized with ECL reagent under the Gel Imaging System (Tannon Science & Technology, Shanghai, China) and protein levels digitized with the Quantity One software (Bio‐Rad).
The data from the study were presented as the mean ± standard error of mean from at least triplicate independent experiments. They were analyzed with one‐way analysis of variance, followed by Fisher's least significant different test (Fisher LSD) and also the independent samples t-test with the Statistical Package for the Social Sciences (SPSS) software (Version 18.0; SPSS, Chicago, IL). Differences were regarded as being significant at P< 0.05.