M7292

Healthy donors with regular estrus cycles were selected for zygote collection. Zygotes were collected using surgical oviduct flushing from donors after superovulation treatment and natural mating. In brief, donors were injected with follicle stimulating hormone (FSH) for estrus synchronization. A total of 60 IU FSH were administered by intramuscular injection in six dosages at 12-h intervals (the first dose was 15 IU and other doses decreased progressively to 5 IU). The animals were then injected with 100 IU of HCG 12 h after the FSH was administered. The females were then subsequently mated with a male rabbit.
Rabbit embryos at the pronuclear stage (approximately 18–20 h post-mating) were transferred into M2 medium (M7167, Sigma, U.S.A.) with a 1% penicillin–streptomycin solution (SV30010, HyClone, U.S.A.) and 10% fetal bovine serum (FBS, SH30084.03, HyClone, U.S.A.). A mixture of in vitro transcribed sgRNA (10 ng/μl) and Cas9 mRNA (40 ng/μl) was injected into the cytoplasm of pronuclear stage embryos. The injected embryos were transferred to M16 medium (M7292, Sigma, U.S.A.) with a 1% penicillin–streptomycin solution and 10% FBS for a 30–60 min culture at 38.5°C, 5% carbon dioxide, and humidity conditions. Then approximately 15−20 injected embryos were transferred into the oviducts of the recipient mother.

Cumulus‐oocyte complexes (COCs) were separate from the large antral follicles of equine chorionic gonadotropin (eCG, Beijing Solarbio Science & Technology, Catalog # P9970)‐primed (46 h) 21‐day‐old female mice, and the cumulus cells of these COCs were removed to obtain denuded oocytes (DOs) as described previously.^[52] DOs got mature in the culture, and then oocyte samples at GV, GVBD, M I, and M II were gathered at the time points of 0, 3, 6, and 14 h during the process of culture for the immunofluorescence (IF) staining of eIF4E1B, microtubules and chromosomes. Ovulated oocytes were obtained from the ampulla of oviducts following a super‐ovulation regimen for mice. Oocytes were collected and cultured in 37 °C pre‐warmed modified eagle medium (MEM) with Earle's salts (Gibco, Catalog # 11095), and added with 75 µg ml⁻¹, 50 µg ml⁻¹, 0.23 mM, and 3 mg ml⁻¹ of penicillin G, streptomycin sulfate, pyruvate, and bovine serum albumin respectively at 37 °C in 5% CO₂ in compressed air and high humidity. Concerning IVF, normal sperms isolated from C57BL/6J adult males were fertilized with ovulated oocytes in vitro. 24 h after IVF, the formation of two‐cell stage embryos transferred into an M16 culture medium (Sigma Aldrich, Catalog #M7292) for subsequent development was scored. Finally, blastocysts were cultured and scored for three‐five days.

Mice at 21-days of age were injected with 5 IU of PMSG and humanely euthanized 44 h later. Oocytes at the GV stage were collected in M2 medium (M7167; Sigma-Aldrich) and cultured in mini-drops of M16 medium (M7292; Sigma-Aldrich) covered with mineral oil (M5310; Sigma-Aldrich) at 37 °C in a 5% CO₂ atmosphere. For microinjection, oocytes were collected in M2 medium with 2.5 μM milrinone to inhibit spontaneous GVBD. mRNAs were transcribed in vitro using a SP6 message machine kit (Invitrogen, AM1450). Microinjection was performed using an Eppendorf microinjector.
For live imaging, mRNAs encoding for RFP-H2B, GFP-tubulin and mCherry-securin were microinjected into WT and DDB1-deleted oocytes and released from milrinone after 2 h. Images of live oocytes were acquired on a DV ELITE High Resolution Invented Living Cell Workstation and imaged at 5 min intervals for 16 h.