Be0046

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.

The H9c2 rat cardiomyocytes were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagles's medium (DMEM; HyClone) containing 1 g/L glucose, 4 mM glutamine, 110 mg/L sodium pyruvate, 10% (v/v) fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin (HyClone) under a humidified atmosphere containing 5% carbon dioxide (CO₂) at 37 °C. To test the effect of inflammatory stimulation on protein O-GlcNAcylation, the H9c2 cells were treated by interleukin (IL)-1β (#HY-P7097A, MCE) and IL-6 (#HY-P7103A, MCE) for 4 or 24 h. To validate this effect, the H9c2 cells were incubated with LMT28 (30 μM; #HY-102084, MCE) or neutralizing antibodies for IL-1β (20 μg/ml; #BE0246, BioXCell) or IL-6 (20 μg/ml; #BE0046, BioXCell) during HA. To attenuate protein O-GlcNAcylation, the H9c2 cells were exposed to 6-Diazo-5-oxo-L-nor-Leucine (DON, 20 μM; MCE) for 24 h.