Using FRET to Define Cdk1-Dependent Ordering of Events During Exit from Second Meiotic M-Phase in Oocytes.

Exit from M-phase requires a precise sequence of molecular events for successful completion, with errors in the process resulting in cell death or aneuploidy, a characteristic feature of cancer and the leading cause of pregnancy failure. Exit from the second meiotic division (MII) in oocytes is a unique event triggered by sperm, involving female anaphase II as well as both male and female pronuclear formation. Very little is known about how these events involving two distinct cell types are coordinated. M-phase exit is driven by inactivation of the master cell-cycle regulator, cyclin-dependent kinase 1 (Cdk1), but details of how Cdk1 orchestrates MII exit has remained sketchy due to technical challenges in studying these events. Here we detail a protocol for undertaking in-depth analysis of Cdk1 activity throughout fertilization in live mouse oocytes using a Cdk1 Fluorescence Resonance Energy Transfer (FRET) biosensor. This protocol illustrates the utility of time-lapse imaging and FRET for interrogating experimentally challenging cell-cycle events.