Fig 4. Sperm behavior during SDF1-induced chemotaxis and contribution of calcium ion.

(A) Image of the chemotaxis slide consisting of two reservoirs connected by a narrow observation area. A time-stable chemical gradient was established by filling the left reservoirs with medium and the right reservoirs with medium containing 1 ng/ml SDF1. A sperm suspension was added to the observation area under a SDF1 gradient. The presence of a time-stable chemical gradient allowed long-term observation of the cellular migratory behavior. (B) Representative video images of sperm movement under a SDF1 concentration gradient with a higher concentration of SDF1 on the right. Note that the sperm showed a turn movement involving asymmetric flagellar bends (yellow arrowhead), changed their swimming direction, and migrated to the right reservoir (high SDF1 concentration). Dotted circles indicate the location of sperm. Time lapse is indicated at the lower right corner of each image. (C) The turn movement rate was evaluated to analyse the chemotactic behaviors of bovine sperm. The sperm movement was recorded by video for 5 min. A turn movement was defined as a sperm movement with a more than 180° turn in the opposite direction. The turn movement rate was calculated as the number of sperm with a turn movement per total migrated sperm. White bars show the rates for sperm that migrated to the left reservoir (low SDF1 concentration), and black bars show those for sperm that migrated to the right reservoir (high SDF1 concentrations). Data are shown as the mean ± SE. n = 3. (D) Relative intracellular Ca²⁺ levels of bovine sperm in the presence or absence of SDF1. The relative fluorescence intensity of the control group was normalised to 1. White bars show the levels for the control group, and black bars show those for the SDF1 group. Data are shown as the mean ± SE (*p<0.05). n = 6. (E) Effect of the Ca²⁺ inhibitor on sperm chemotaxis to SDF1. Sperm chemotaxis was evaluated by a chemotaxis chamber technique in the same manner. NNC was added to both chambers at a final concentration of 10 μM. White bars show the levels for the control group, and black bars show those for the NNC group. Data are shown as the mean ± SE (*p<0.05). n = 4. (F) Proposed mechanism of SDF1-induced chemotaxis for bovine sperm. The results suggest bovine sperm chemotaxis toward SDF1 is regulated as follows: (i) external Ca²⁺ uptake via Ca²⁺ channels, (ii) a rise in the intracellular Ca²⁺ concentration, (iii) asymmetric flagellar bends, and (iv) modulation of the swimming direction using the turn movement.