Animal experimental procedures in our study were all approved by the Animal Ethics Committee of Nanjing Medical University (NJMU) (approval no. IACUC-1809011), and all mice were housed in the Animal Core Facility (ACF) of NJMU under standard specific pathogen free (SPF) conditions of ACF.
Three-week-old SCID (Severe Combined Immunodeficient) mice were bought from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China) and housed in the ACF.
Three-week-old control CD1/ICR mice were bought from ACF.
Primary antibodies: Anti Beta Actin mAb (Cat#:S6007M, Bioworld, Dublin, OH, USA); Rabbit anti OMPC (Cat#: bs20213R, Bioss Antibodies, Beijin, China); Acetyl-Histone H3(K27)(D5E4) XP(R) rabbit mAb (Cat#: 8173s, Cell Signaling Technology, Danvers, MA, USA); Histone H3 (Trl-Methyl K4) pAb (Cat#: BS7232, Bioworld, Dublin, OH, USA); CR2 rabbit pAb (Cat#: A8407, ABclonal, Wuhan, Hubei, China); CD46 rabbit pAb(Cat#: A1653, ABclonal, Wuhan, Hubei, China); FCGR1A pAb (Cat#: BS6238, Bioworld, Dublin, OH, USA); Phospho-Histone H3(Ser 10) Mouse McAb (Cat#: 66863-1-Ig; Proteintech, Rosemont, IL, USA); Tubulin α (G436) mAb (Cat#: BS1699M; Bioworld, Dublin, OH, USA); Anti-RELA (Phospho-Ser536) rabbit polyclonal antibody (Cat#: D155006; BBI, Shanghai, China), AffiniPure Goat anti-Mouse IgG, light chain specific (Jackson ImmunoResearch Laboratory, Cat#: 115-005-174, West Grove, PA, USA).
Secondary antibodies: Horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG and HRP-conjugated goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratory (West Grove, PA, USA). Goat anti-mouse IgA-FITC (Bersee, Cat#: BFR522, Beijing, China); Goat anti-mouse IgM-FITC (Bersee, Cat#: BFR523, Beijing, China); Cy2-conjugated donkey anti-mouse IgG (Code: 715-225-150), Cy3-conjugated donkey anti-mouse IgG (Code: 715-165-150), all purchased from Jackson ImmunoResearch Laboratory (West Grove, PA, USA).
Oocyte Collection and In Vitro Culture
Fully-grown GV oocytes were collected from three-week-old female control or SCID mice. Oocytes were released by puncturing follicles with a sterile syringe needle in a MEM+ medium (0.01 mM EDTA, 0.23 mM Na-pyruvate, 0.2 mM penicillin/streptomycin and 3 mg/ml BSA in MEM). After the cumulus cells from the cumulus-oocyte complexes (COC) were washed off, cumulus-free oocytes were cultured in 100 µl mini-drops of MEM+ containing 10% fetal bovine serum (FBS) (Thermo Fisher) covered with mineral oil at 37.0 °C in an incubator with 5% O2, 5% CO2, and a humidified atmosphere.
Cell Culture and mitotic index assay
NIH3T3 cells were from ATCC (Cat No.: CRL-1658) and sold by Procell Life Science & Technology Co. (Wuhan, Hubei, China). The cells were cultured in DMEM with 10% FBS. For mitotic index assay, NIH3T3 cells were fixed and immunostained for tubulin and p-H3, the cells with strong p-H3 staining on the chromosomes are mitotic cells. For each sample, at least 1000 cells were counted. Mitotic index is the number of mitotic cells divided by the number of total counted cells.
Immunofluorescence Staining of Oocytes and Image Taking
Two distinct protocals were used for the permeation & fixation of oocytes. The blocking, primary antibody incubation, secondary antibody incubation and oocyte mounting procedures after permeation & fixation are the same for these two protocals.
Permeation & fixation：
1). For experiments in Fig. 7E-G, we wanted to maintain IgG signals furthest on the membrane. After three times of quick wash in PBS/0.05% PVP (polyvinylpyrrolidone), oocytes were first fixed in 3.7% FPA in PHEM for 30 minutes at room temperature and washed with PBS/PVP three times at 10 minutes each. Then oocytes were permeated in 1% Triton X-100/PHEM (60 mM PIPES, 25 mM Hepes, pH 6.9, 10 mM EGTA, 8 mM MgSO4) for 10 minutes.
2). For all other experiments except Fig. 7E-G, we wanted to get best quality of cytoplasmic signal. After three times of quick wash in PBS/PVP, oocytes were first permeabilized with 0.5 % Triton X-100/PHEM (60 mM PIPES, 25 mM Hepes, pH 6.9, 10 mM EGTA, 8 mM MgSO4) for 5 minutes. After washed with PBS/PVP three times at 10 minutes each, oocytes were fixed in 3.7% FPA in PHEM for 20 minutes at room temperature.
Blocking, primary antibody incubation, secondary antibody incubation and oocyte mounting:
After being washed with PBS/0.05% PVP (polyvinylpyrrolidone) three times at 10 minutes each, oocytes were blocked in blocking buffer (100 mM glycine and 1% BSA in PBS) for one hour at room temperature. Primary antibodies were then diluted in blocking buffer and oocytes were incubated in it overnight at 4.0 °C. Fluorescent secondary antibodies (Jackson ImmunoResearch Laboratories) were used at 7.5 μg / ml. DNA was stained with 0.3 μg / ml Hoechst 33258. Next, buffers were were slowly and gentely removed from around the oocytes, which immobilized the oocytes on the slide; then a drop (about 5-10 μl) of anti-fade solution (0.25% n-propyl gallate and 90% glycerol in PBS) was mounted onto the oocytes and oocytes were covered by a coverglass. To avoid the deformation of the oocytes, a double-stick tap was pre-placed between the slides and coverslips. Specimens were imaged with IQ2 on an Andor Revolution spinning-disk confocal system (Andor Technology PLC, Belfast, UK) mounted on an inverted TiE microscope (Nikon, Japan) with a 60 ×, 1.4 NA objective and captured with cold CCD camera (Andor). Most images are displayed as maximum intensity projections of the captured z stacks.
Immunoprecipitaion, commassie staining and mass-spec
2.5 μg HRP-conjugated goat anti-mouse IgG antibody was first coupled to 30 μl protein-A/G beads (Yeasen, Shanghai, China) for 4 hours at 4°C on a rotating wheel in 250 μl immunoprecipitate (IP) buffer (20 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1 mM EGTA, 150 mM NaCl, 0.05% Triton X-100, 0.05% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) with 1:100 protease inhibitor (Sigma) and 1:500 phosphatase inhibitor (Sigma). Meanwhile, 400 oocytes were lysed and ultrasonicated in 250 µl IP buffer and then pre-cleaned with 30 µl protein-A/G beads for 4 hours at 4 °C. After that, protein A/G-coupled Rabbit IgG or specific antibody were incubated overnight at 4 °C with pre-cleaned oocyte lysate supernatant. Finally, after being washed three times (10 minutes each with 250 µl IP buffer), the resulting beads with bound immunocomplexes were subjected to SDS-PAGE and commassie staining. The gel band corresponding to the band from IgG blot was cut out and sent to Biotech-pack (Beijing, China) for mass spec identification of proteins.
H3K27ac and H3K4me3 CHIP-seq were perform with Hyperactive In-Situ ChIP Library Prep Kit under Targets and Tagmentation (CUT&Tag) technology (Vazyme) according the manufacturer's instruction. The kit using a hyperactive Tn5 transposase to precisely binds the DNA sequence near the target protein. In brief, lysate of 100 oocyte were incubated with ConA beads, then primary and secondary antibody were in turn incubated with oocyte lysate, next Hyperactive pG-Tn5/pA-Tn5 Transposon was incubated with the sample and tagmentation were performed. Finally DNA was extracted and library was prepared as instructed, and DNA library was sent to Novogene (Beijing, China) for sequencing.
Virus SSRNA production and microinjection
Immunity-inducing GU-rich virus ssRNA (Single-strand RNA) sequences were according to reported (25). We selected two sequences: 5'-GACTTGAGCGAGCGCTTTTTT-3' (ssRNA-6T) and 5'-GTCCGGGCAGGTCTACTTTTT-3' (ssRNA-5T). SsRNA were produced and purified using the T7 Ribomax Express RNAi System (Promega) according to the manufacturer’s instructions, then aliquoted and stored at -80°C. The difference here is that the RNA is single-stranded from one transcription reaction. Primers for DNA templates are in suppl. table 2.
SsRNA was adjusted to 5uM with nuclease-free water, and about 10 pl of ssRNA was injected into GV oocytes with a programmed microinjector. 10 μM milrinone was included to prevent GVBD.
LPS, CSF1, H2O2, NMN and SC75741 treatment
LPS (Cat#: L2880, Sigma, St. Louis, MO, USA) was added into oocyte IVM medium at a final conc. of 10 EU/ml for 16 hour; CSF1 (Cat#: HZ-1207, Proteintech, Rosemont, IL, USA) was added into oocyte IVM medium at a final conc. of 10 ng/ml for 16 hour; H2O2 (Cat#: 88597, Sigma, St. Louis, MO, USA) was added into oocyte IVM medium at a final conc. of 30 mM for 30 min; NMN (Cat#: S5259, Selleck Chemicals, Houston, TX, USA) was added into oocyte IVM medium at a final conc. of 1 mM for 16 hour; SC75741 (Cat#: S7273, Selleck Chemicals, Houston, TX, USA) was added into oocyte IVM medium at a final conc. of 0.1 μM for 16 hour.
E-coli fluorecence labeling and treating oocytes with E-coli
FICT-d-Lys（Cat#: I0201,Bioluminor, XiaMen, Fujian, China）was added into LB medium at a final conc. of 0.1mM. 2.5 μl DH5α competent E.coli (Cat#: C502, Vazyme, Nanjing, Jiangsu, China) were added into 1 ml LB medium and the E.coli culture were grown at 37 °C until OD600 reached 0.6. Then E.coli was collected and washed by PBS, then re-suspend 1ml PBS. Finally, 10 μl PBS / with Ecoli was added into 100 μl IVM medium with 50 oocytes during the whole IVM period. Except for maturation assay done at 16 hour of E.coli treatment, all other E.coli-treated oocyte experiment were done at 6 hour.
Antibodies for CR2, CD46 and FCG1R were added into oocyte IVM medium at a final conc. of 0.05 mg/ml to inhibit corresponding receptor on the oocyte membrane. 0.065 mg/ml antibody for IgG light chain was microinjected into GV oocytes (10m pl per oocyte) to inhibit IgG. To remove the toxic preservative within the commercial antibodies, the antibodies were buffer-exchanged into PBS/50% glycerol with a size-exclusion spin column until the original preservative-containing antibody solution was less than 10-4 of the final solution.
Oocyte cDNA Synthesis and Amplification of IgG VDJ region
RNA extraction and cDNA synthesis were performed with NEBNext® Single Cell/Low Input cDNA Synthesis& Amplification Module (Cat No.: E6421, NEB, Lpswich, MA, USA) according to the instruction from 100 oocytes. For IgG VDJ amplification, two round of PCR were done with two set of primers respectively. The primer sequences are in supplementary table 1. The DNA polymerase was Phanta high-fidelity DNA polymerase (Cat No.: P505-d1, Vazyme, Nanjing, Jiangsu, China). Then PCR products were ligated into cloning vector (Vazyme, Cat No.: C601-02) and transformed into competent DH5α, and 50 colonies (ligated cDNA from each colony corresponds to the mRNA of a single oocyte) were sent to GENEWIZ (Suzhou, Jiangsu, China) for sequencing.
Detection of ROS Generation
The ROS Assay Kit (Cat#: S0033, Beyotime, Beijing, China) was used to detect ROS generation in oocytes. In brief, oocytes were incubated with a dichlorofluorescein diacetate (DCFH-DA) probe for 20 min at 37 °C in the dark, washed, and mounted on slides for confocal imaging.
Mitochondrial staining and ATP measurements
For mitochondrial staining, oocytes were incubated in Hepes containing 100 nM Mito Tracker (Cat#: M7521, Invitrogen, Carlsbad, CA, USA) and 10 µg/ml Hochest 33342 (Sigma) for 30 min. Images were taken with an Andor Revolution spin disk confocal workstation.
For ATP measurements, 15 oocytes were first lysed with 100 µl ATP lysis solution (Cat#: S0026, Beyotime) on ice. The samples were then detected by enzyme-labeled instrument Synergy2 (BioTek, Winooski, VT, USA) to evaluate ATP level.
Apoptosis was detected with an Annexin V-FITC/PI Apoptosis Detection Kit (Cat#: 40302ES20, YEASEN). Oocytes were stained with 5 μl annexin V-FITC and 10 μL PI Staining Solution diluted in 100 μl binding buffer for 15 min in the dark at RT. After washing, oocytes were mounted onto glass slides, and images were obtained as above.
Animal / oocyte inclusion, experiment grouping, data collection, and data analysis
Any selected oocyte must be of normal quality (fully-grown oocyte from an antral follicle, normal diameter, tight connection between zona pellucida and oocyte membrane, etc). Any selected female mouse has to be physically healthy (normal body weight, eats normally, normal activity, etc). Any oocyte or mouse of bad quality or unhealthy will be excluded.
For sample size in oocyte-related experiment, about 3-5 oocytes were randomly selected for one repeat and thus about 9-15 oocytes were analyzed for three independent repeats. We found that measurements from 9-15 oocytes could form a normal distribution and are fairly reliable for a statistic analysis. For sample size in mouse-related experiment, similarly, about 3-5 mice were randomly selected for one repeat and 9-15 mice were analyzed for three independent repeats.
We tried to follow the double-blinding roles for both mouse and oocyte-related experiment grouping, data collection, and data analysis. For each independent repeat, oocytes were obtained from multiple mice to eliminate natural differences between individuals; oocytes were randomly divided into different experimental groups. About 50 oocytes per group for immunofluorescence / live fluorescence or 100 oocytes per group for western blot were used. For image taking, the order for different groups was solely random. And within the same group, oocytes were again randomly selected for image taking (We didn't take images from all oocytes). The index setting for image taking was always identical for each group of the same experiments. The order of different groups for data measurement is also random. All intensity measurements were done with Image J on the original tif file without any adjustment of brightness/contrast. Fluorescence intensity was always a net intensity obtained by a measured image intensity subtracted by the adjacent background intensity. For band quantification in western blot, an integrated intensity was obtained by the average intensity multiplied by the band area; the average intensity was again obtained by measured image intensity subtracted by the adjacent background intensity.
All experiments were repeated at least three times. Data are presented as mean ± SEM. Comparisons between two groups were made by Student’s t test. Differences among more than two groups were compared using one-way ANOVA. P < 0.05 was considered statistically significant. Statistical analyses were conducted with GraphPad Prism.