1. Generation of Gdf9-Cre;mTmG mice:
(1) To generate the Gdf9-Cre;mTmG mouse models, adult healthy Gdf9-Cre males were crossed with mT/mG female mouse in 1:2.
(2) DNA of mouse tail extracted and genotyped to identify the Gdf9-Cre;mTmG female before postnatal day (PD) 7.
Gdf9-Cre primers: TCTGATGAAGTCAGGAAGAACC
mT/mG primers: CTCTGCTGCCTCCTGGCTTCT
2. Preparation of culture medium:
Medium α (MEMα; Gibco) was supplied with 10% FBS (Gibco), 1% insulin-transferrin-selenium (ITS; Sigma-Aldrich) and 0.1% penicillin and streptomycin (Thermo Fisher Scientific), and was pre-warmed to 37℃ before using.
3. Collecting the denuded oocytes:
(1) Ovary was collected from Gdf9-Cre;mTmG females at PD 35.
(2) Remove the ovarian bursa carefully by sterile 1 mL syringe in cold PBS buffer.
(3) By tearing the ovaries with Omnican U-40 insulin syringe, denuded oocytes at GV stages were collected in the culture medium. Only the healthy oocytes with an average diameter around 65 μm were collected for the further experiments.
(4) Washing the collected oocytes three times with ~50 μL culture medium microdroplet by mouth pipette gently.
*Tips: Transferring the oocytes gently to guarantee the integrity of ZP.
4. Imaging the cellular membrane structure on living oocytes:
(1) Transfer around 10 oocytes to the culture medium microdroplet (~20 μL) in 35 mm glass bottom dish.
(2) Covered the microdroplet with 1.5 mL mineral oil (Sigma-Aldrich).
(3) Photographed in a living cell workstation (Okolab) at 37°C, 5% CO2 by Andor Dragonfly 502 spinning-disc confocal with following index. In details, images were acquired by an Andor Dragonfly 502 spinning-disc confocal microscope equipped with a 63×, 1.40 N.A. oil objective (Leica HC PL APO), a scientific complementary metal-oxide semiconductor (sCMOS) camera (Andor Zyla 4.2), and 488-nm (mG) and 568-nm (mT) lines of the Andor Integrated Laser Engine (ILE) system with a spinning-disc confocal scan head (Andor Dragonfly 500). The oocytes were acquired through z-step mode with the index. In detail, images were acquired with laser 488-nm around 10-20%, laser 568-nm around 15-25%, exposure time 100-200 ms and Z-step 0.5-0.7 μm for 25-35 μm by Fusion 2.1 software.
(4) After acquisition, videos or single time-point images were processed by ImageJ (http://rsbweb.nih.gov/ij/) for projection of all z-stacks and merged color channels.
(5) To finally detect the Oo-Mv on oocyte, the rotary 3D video was processed by Imaris (https://imaris.oxinst.com/) software.
*Tips: The index of imaging might be changed with the situations of microscope such as the environment temperature and humidity.