Isolation and Culture of Mouse Oocytes

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.