Fig 1. Evaluation of sperm migration toward a COC using 12-well dishes.

(A) Image of a 12-well dish for the sperm migration assay. (B) Schematic illustration with both top and side views of a 12-well dish arranging a single COC and sperm. Note that the 12-well dishes were filled with medium and a single mature COC and 50 μl of sperm suspension were deliberately placed in the specified wells of the dishes as shown in this illustration so that sperm were free to swim around in the dish and go back and forth between adjacent wells. These cells were cultured for 3 h under mineral oil at 38.5°C with 5% CO₂. (C) Representative images of COC-containing and empty wells in the presence or absence of AMD3100 after coincubation for 3 h. Yellow and black arrowheads indicate sperm aggregation and a single COC, respectively. Bars = 500 μm. (D) Comparison of the number of sperm that migrated to COC-containing and empty wells in the presence or absence of AMD3100 after three hours of incubation. Schematic illustrations on the right of each graph indicate the experimental design of the 12-well dishes. White bars show the numbers for the COC-containing well, and black bars show those for the empty well. The number of oocytes examined in this experiment is 28 for control group and 21 for AMD3100 group. Data are shown as the mean ± SE. Different letters indicate a significant difference (p<0.05). This experiment was repeated six times.