Oocyte Maturation Dynamics via mRNA Microinjection

Recombinant mRNA for microinjection was prepared by in vitro transcription of 500 ng of linearized mCHERRY or ELAVL2°-mCHERRY plasmids using the mMESSAGE kit (Ambion) according to the manufacturer’s instructions. Recombinant mRNA was polyadenylated using ATP-polyA tailing kit (Ambion) and purified by RNeasy mini kit columns (Qiagen). Fully grown GV oocytes for microinjection were obtained from PMSG-primed H2B-GFP mice⁶³ (link) backcrossed to CD1 strain. Oocytes were microinjected with approximately 5 pl of in vitro transcribed mRNA (336 or 112 µg/ml) and cultured for 2 h in OptiMEM medium supplemented with 10% FCS (Invitrogen) containing 2.5 µM milrinone (Sigma) that reversibly blocks maturation of oocytes. After 2 h, oocytes were washed in milrinone-free medium and let to mature for 18.5 h. Leica TSC SP5 (Leica Microsystems) equipped with an HCX PL Apo Lambda Blue 40× 1.25 oil objective was used for time-lapse confocal microscopy. Images(12 z-confocal sections every 7.4 µm, 1024 × 1024 xy pixel resolution, 16 bit depth) were acquired every 10 min for 18 h. Imaging started 40 min after milrinone removal.