Spinning disc confocal scanner

WT and Skp1^cKO (Skp1^f/− Ddx4-Cre) female mice (3 to 4 weeks old) were hormonally primed with 5 U of PMSG (pregnant mare’s serum gonadotropin) (catalog number 367222, Calbiochem) 48 hours before oocyte collection. GV-intact oocytes were collected in bicarbonate-free minimal essential medium with polyvinylpyrrolidone and Hepes (MEM-PVP) (48 ), denuded from cumulus cells, and cultured in Chatot-Ziomek-Bavister (CZB) medium (49 (link)) covered with mineral oil (catalog number M5310, Sigma) in a humidified atmosphere of 5% CO₂ at 37°C. Meiotic resumption was inhibited by addition of 2.5 μM milrinone. Milrinone was subsequently washed out to allow meiotic resumption 1.5 hours after collection. Oocytes were checked for nuclear envelope breakdown (GVBD) 1.5 hours after washout, and those that did not enter GVBD were removed from the culture. Confocal images were collected with a microscope (DMI4000 B, Leica) equipped with a 63× 1.3 numerical aperture (NA) glycerol immersion objective lens, an xy piezo Z stage (Applied Scientific Instrumentation), a spinning disc confocal scanner (Yokogawa Electric Corporation), an electron multiplier charge-coupled device camera (ImageEM C9100-13; Hamamatsu Photonics), and an LMM5 laser merge module with 488- and 593-nm diode lasers (Spectral Applied Research) controlled by MetaMorph software (Molecular Devices).