Fig. 4.

Study of the hyperactive motility and acrosome reaction calcium downstream KOR in human spermatozoa. A,, Study of the hyperactive motility by 1 min co-incubation of U50488H (1 μm) and: NNC-55–0395 (10 μm), the Catsper inhibitor; Mibefradil (30 μm), a calcium channel activator and U73122 (3 μm), the PLC inhibitor. X axis shows the different treatments used for this study and the Y axis represents the normalized data of the % of motile spermatozoa. The normalization was performed using the untreated samples. n, = 6. (*p, < 0.05 and **p, < 0.01 versus, Control; +p, < 0.05 versus, Mibefradil). B,, Study of the acrosome reaction by the 60 min co-incubation of U50488H (1 μm) and: NNC-55–0395 (10 μm), Mibefradil (30 μm) and U733122 (3 μm). X axis shows the different treatments used for this study and the Y axis represents the normalized data of the % of acrosome reacted spermatozoa. The normalization was performed using the untreated samples and the acrosome reacted samples. The progesterone was used as the positive control of the acrosome reaction. n, = 7. (*p, < 0.05 and **p, < 0.01 versus, Control; +p, < 0.05 versus, Mibefradil). C,, Intracellular free Ca²⁺ measurements in human sperm cells loaded with Fura-2 in response to the solvent of U50488H (H₂O) (control samples) and U50488H (1 μm). Subsequent addition of 30 μm Mibefradil as a calcium channel activator and 1 μm progesterone to the same sperm aliquots. The progesterone caused a typical biphasic [Ca²⁺]i progesterone response. The X axis shows time in seconds and the Y axis shows [Ca²⁺]i expressed by Fura-2 ratio variation. Calibration of [Ca²⁺]i was achieved adding Triton X-100 (TX), to obtain the maximal response, followed by addition of EGTA to obtain the minimal response. n, = 5. AR = Acrosome reacted. HA = Hyperactive.