Assessment of cell apoptosis by Hoechst 33342 staining

Hoechst staining was used to assess MGCs and CCs apoptosis because previous studies have shown that TUNEL and Hoechst staining are comparable methods for detecting apoptosis⁶⁰. The MGCs obtained as described above and CCs released from 60–80 oocytes were dispersed by repeatedly pipetting using a thin pipette. The dispersed cells were centrifuged (200 × g) for 5 min at room temperature. The pellets were resuspended in 50 μl of M2 medium containing 10 μg/ml of Hoechst 33342 and stained in the dark for 5 min. The stained cells were resuspended in M2 and centrifuged at 200 × g twice (5 min each). Finally, a 5-μl drop of suspension was smeared on a slide and examined under a Leica DMLB fluorescence microscope (400×). Six to eight fields were randomly observed on each smear, and the percentages of apoptotic cells were calculated double blindly by 2 investigators.