Mr 106 d

Zygotes were collected from the ampulla and cumulus cells removed by incubation with 300 μg/mL hyaluronidase (H4272, Sigma) in M2 medium (M7167, Sigma). After washing the embryos in a few drops of KSOM medium (MR-106-D, Millipore), they were cultured in KSOM microdrops under mineral oil (NO-400K, Nidacon) in an incubator with 5% CO₂ at 37°C. Embryos were handled with a mouth aspirator (A5177-5EA, Sigma) coupled to fire-polished glass Pasteur pipettes and collected at different stages of development from the in vitro cultures either for RNA-seq or protein immunostaining as detailed in the sections below. To track their development in control or IL-6 blocking conditions, phase contrast pictures of the developing embryos were taken with a Leica inverted microscope (DMI6000B) and embryos were counted using Fiji software.
The amounts of IL-6 produced by the various samples was assessed using an ELISA kit (M6000B, R&D Systems) according to the manufacturers’ instructions. Emissions of the samples at 450 nm wavelength were measured in a plate reader (SPECTROstar Nano, BMG Labtech). Anti-IL-6 blocking treatments were performed by adding 0.1 mg/mL of antibody (BE0046, BioXCell) IgG1 or anti-horseradish peroxidase antibody (BE0088, BioXCell) IgG1 as a control in culture microdrops from the zygote stage up to late blastocysts.

Microinjection of mouse embryo was performed referring to our previously described method.²² Briefly, superovulated B6D2F1 female mice were mated with adult B6D2F1 males, and zygotes were collected from female oviducts at 20 h post-human chorionic gonadotropin (hCG; Sansheng, China) injection. pCMV-PE2 plasmid (Addgene; 132775) (100 ng/μL), pegRNA (50 ng/μL), and nicking sgRNA expression plasmids (50 ng/μL) were mixed, and 2−4 pL mixture was injected to the cytoplasm of zygotes at 21−24 h post-hCG. Microinjection was performed in a droplet of M2 (Sigma-Aldrich, USA; M7167) containing 5 μg/mL cytochalasin B by using a Piezo-driven micromanipulator (Prime Tech, Japan; Pmm4G). Then the embryos were cultured in potassium simplex optimized medium (KSOM; Millipore, USA; MR-106-D) at 37°C in 5% CO₂ atmosphere. The embryos were cultured until blastocyst stage (E4.5) and then subjected to genotyping. To generate live pumps with targeted genome editing by a PE3 device, embryos were transferred to the oviducts of pseudopregnant ICR mice immediately after microinjection, with 20 embryos for each surrogate.

Animal care and experiments were conducted in accordance with the Guidelines of Animal Experimentation of the National Institutes for Natural Sciences. All animal experiments were approved by the Animal Research Committee of the National Institutes for Natural Sciences. Mice were maintained in a light- and temperature-controlled room using a 12 h light:12 h dark cycle at 23 ± 2°C.
Males of R26-ZO-1EGFP (Katsunuma et al., 2016 (link)) or wild-type ICR (Japan CREA) were mated with wild-type ICR females to obtain blastocysts. Preimplantation E3.5 embryos were flushed from uteri with KSOM (MR-106D, Millipore). Embryos were cultured in KSOM covered with mineral oil at 37°C and 5% CO₂. Fluorescent signals were monitored using a confocal microscope (A1, Nikon) or a spinning disc confocal microscope (CV1000, Yokogawa). For immunofluorescence, embryos were fixed at 4°C in 4% paraformaldehyde (PFA) in PBS overnight. For ouabain treatment, 10 mM ouabain (cat. # O3125, Sigma-Aldrich) in DMSO was diluted to the indicated concentrations.