M16 media

Mouse embryonic stem cells were derived from the Ie mouse and maintained in 2i media as previously described [17 (link)]. E2.5 embryos were flushed from oviducts in M2 media (Sigma) and maintained overnight in M16 media (Sigma-Aldrich®, MA, USA) supplemented with 1 µM PD0325901, 3 µM CHIR99021, and 1× penicillin/streptomycin. Subsequently, blastocysts were maintained in 2i media for two days followed by isolation of inner cell mass by immunosurgery. The inner cell mass was maintained in 2i media for 5–7 days until cell outgrowths were dissociated and expanded following standard cell culture procedures.

Animals were housed in filter-top cages in a specific pathogen-free environment and fed a standard diet. All work involving animals complied with all relevant ethical regulations and was approved by the Animal Ethics Committee at the University of Queensland. Mice were allowed ad libitum access to food and water and maintained in a facility with a 12 h light/dark cycle and a 50% relative humidity at 22–24 °C. Ovaries were isolated from 3 to 4-week-old B6CBAF1 female mice 44–46 h following intra-peritoneal injection of 7.5 international units (IU) of pregnant mare’s serum gonadotrophin (PMSG; Pacificvet). Dissected ovaries were transferred to the lab in pre-warmed αMEM HEPES-buffered medium (Sigma-Aldrich) containing 50 μM 3-isobutyl-1-methylxanthine (IBMX; Sigma-Aldrich), which prevents oocytes from undergoing GVBD^{7 (link),50 (link),51 (link)}. Ovaries were punctured in IBMX-treated αMEM HEPES-buffered medium in 35 × 10 mm dishes using a 27 G needle under direct vision on the stage of a stereomicroscope (M165C, Leica Microsystems). Only fully grown cumulus-covered oocytes were isolated, denuded by mouth pipette and used for further experiments. For longer term culture and for all confocal imaging, oocytes were cultured in micro-drops of M16 media (Sigma-Aldrich) under embryo-tested light mineral oil (Sigma-Aldrich) at 37 °C in an atmosphere of 5% CO₂ in air.

Four week-old ICR mice were sacrificed by cervical dislocation 48 h after an injection of pregnant mare's serum gonadotropin (PMSG, 5IU/mouse). Cumulus oocyte complexes (COCs) were collected by puncturing the ovaries in M2 media (Sigma-Aldrich, St. Louis, MO, USA). Each mouse generally ovulates about 17–20 COCs. Cumulus cells were mechanically removed from the oocytes by a mouth-controlled pipette. Collected oocytes were cultured in M16 media (Sigma-Aldrich) for 3 h (prometaphase I, PMI), 8 h (metaphase I, MI), or 12–16 h (metaphase II, polar body) in 37°C, 5% CO₂ incubator in a drop of M16 media covered with mineral oil [25] (link). In some experiments, the oocytes were treated with nocodazole (10 μg/ml), taxol (1 μM) or cytochalasin D (cytoD, 10 μM, Sigma-Aldrich) by adding it to the culture media. Naturally mated female mice were sacrificed on day 2, and oviducts were flushed to obtain 2-cell stage embryos. Uteri from day 4 pregnant mice were flushed to obtain blastocysts. Embryos were cultured in KSOM-AA (Millipore, Billerica, MA, USA). Embryos were treated with nocodazole (3 μg/ml) for 12 h (Schuh and Ellenberg, 2007) and recovered in media for 40 min before they were processed for immunofluorescence staining.

All the prerequisites were performed. Maximum eggs (oocytes) were extracted from each mouse, collected in M2 medium plates (Figure 1A). The oocytes were incubated at 37 °C for 2–4 h in the M16 media (Sigma, USA). Similarly, the plasmid vectors were directly injected into the pronuclei of each oocyte following the standard protocol (Figure 1B). After injection, the oocytes were incubated for 4–6 h at 37 °C and were transferred directly to the pseudo-pregnant CD1 females (Figure 1C). Olympus IX71 inverted microscope, and Narishige microinjector was used.
Bex3 specific primers targeting the upstream and downstream of sgRNAs were designed manually. The PCR was carried out according to the prescribed protocol (Oo et al., 2020 (link)). GelDoc was used to observe the gel pictures (Malumbres et al., 1997 (link)). The transgenic littermates were separated accordingly. The chimaeras were obtained by crossing the littermates.

Human chorionic gonadotropin (hCG) and PMSG were purchased from Ningbo Sansheng Pharmaceutical Co. (China). M16 media and hyaluronidase were obtained from Sigma-Aldrich (Germany). The anti-fade reagent with DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) was supplied by Life Technologies Corp. (Carlsbad, CA, USA).
The antibody against Rps26 was obtained from Proteintech Group, Inc., while antibodies against H3K4me3, H3K9me3, RNA polymerase II, p-RNA polymerase II (p-S2), Gdf9, Bmp15, and Cx37 were purchased from Abcam. Antibodies against p-Akt (Ser473), Akt, p-Rps6 (Ser235/236), Rps6, p-Foxo3a (Ser253), Foxo3a, and actin were purchased from Cell Signaling Technology (Beverly, MA, USA). The antibody against P27 was purchased from Santa Cruz Biotechnology Inc., and the antibody against 5mC was purchased from Calbiochem Co. The Click-iT® RNA Alexa Fluor® 594 imaging kit was obtained from Invitrogen Co. Ltd.

The Animal Research Committee of Nanjing Agricultural University in China authorized the study and our operations with mice followed the requirements (Su-XYXK-2017-011). Female 4-week-old ICR mice were kept at a constant temperature of 24 °C in a 12-h light-dark cycle and had unrestricted access to food and water during the research period. After the experiments, all mice were killed by cervical dislocation. We collected germinal vesicle intact oocytes from mouse ovaries in M2 medium (Sigma, St. Louis, MO, USA), washed them three times, and cultured the oocytes in M16 media (Sigma, St. Louis, MO, USA) for 8 h (metaphase I, MI) or 12 h (metaphase II, MII) under paraffin oil at 37 °C in a 5% CO₂ environment.