Ca free ksom

Confocal microscopy was performed on a Leica SP5 resonant scanner confocal microscope (Biological Imaging Facility, Northwestern University) and a Zeiss LSM 510 Confocal Microscope (Quantitative Bioelemental Imaging Center, Northwestern University). Intracellular zinc was visualized using 50 nM ZincBY-1 and extracellular zinc was visualized using 50 µM FluoZin-3 (Life Technologies). DNA was visualized using 10 µg/mL Hoechst 33342. In figures, fluorescence images within the same panel were collected and displayed using the same parameters. In activation experiments, eggs were activated using 10 mM SrCl₂ in Ca-free KSOM (Millipore). Further experimental details are provided in the Supplementary Methods.

GV-stage oocytes were isolated from cumulus–oocyte complexes (COCs) collected from the ovaries of sexually mature (6–10 weeks old) female CD-1 mice injected with 5 IU pregnant mare’s serum gonadotropin (PMSG, Sigma-Aldrich) 48 h before sample collection. Collection was performed in Leibovitz’s L-15 medium (Life Technologies) supplemented with 1% (v/v) FBS (Life Technologies) and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX, Sigma-Aldrich). To collect MII-arrested eggs at the metaphase II (MII) stage, females were injected with 5 IU PMSG and then 5 IU human chorionic gonadotropin (hCG, Sigma- Aldrich) 46 h later. Eggs were isolated from the oviducts 14 h after administration of hCG. Cumulus cells were denuded using 0.3% (w/v) hyaluronidase. Parthenotes were obtained by activating MII eggs in 10 mM SrCl₂ in Ca-free KSOM (Millipore) for 3h at 37 °C in an atmosphere of 5% CO₂. Successfully activated cells were selected based on extrusion of the second polar body. Animals were treated in accord with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals. Food and water were given ad libitum. The Northwestern University Institutional Animal Care and Use Committee (IACUC) approved all protocols.