In Vitro Oocyte Maturation Assay

Oocyte in vitro maturation assays were performed as previously described (Brunet et al., 2008 (link)). Briefly, oocytes were collected from ovaries of 10- or 26-week-old hmmr^+/+ and hmmr^m/m mice and placed in M2 medium pre-warmed to 37°C and supplemented with 4 mg/ml BSA and 1 mM milrinone. For video microscopy of oocyte meiotic maturation, oocytes were transferred to a Ludin Chamber containing M2 medium with 4 mg/ml BSA. Time-lapse images were acquired using a Photometrics CCD camera (CoolSnap HQ2) mounted on a Leica HC PL APO 20×/0.7 NA objective enclosed in a thermostatic chamber (Life Imaging Service). Images were taken every 15 min for 18–20 h at 20× magnification. Metamorph 7.0 (Universal Imaging) and ImageJ (NIH) software were used for image analysis.