Mouse sperm acrosome exocytosis

CD1 male mice (10 to 12 weeks old) were maintained in a 12-hour light and 12-hour dark cycle, at 23°C and 55±15% humidity, with water being always accessible. Animals were euthanized and cauda epididymal sperm collection followed. Cauda epididymis was cut at multiple sites, placed in 500 µl of non-capacitating medium (NC) and incubated at 37°C for 15 min. Supernatant was collected and pre-incubation with 100 nM SiR-actin in NC of recovered sperm took place for 10 min. Once dyed, sperm were once more incubated for at 37°C for 60 min in capacitating (CAP) conditions. NC used was a modified TYH medium (119.3 mM NaCl, 4.7 mM KCl, 1.71 mM CaCl₂•2H₂O, 1.2 mM KH₂PO₄, 1.2 mM MgSO_4•7H₂O, 0.51 mM sodium pyruvate, 5.56 mM glucose, 20 mM HEPES and 10 µg/ml gentamicin). For CAP conditions, 5 mg/ml BSA and 15 mM NaHCO₃ were added. Sperm were immobilized in coverslips treated with concanavalin-A (1 mg/ml). The imaging chamber was loaded with NC with 0.5 µM FM4-64 and 100 nM SiR-actin. Dye excitation was provided by 561 nm and 640 nm lasers. Using a NanoImager-S microscope (ONI, Oxford Nanoimaging Ltd) equipped with a 100X, 1.4 NA, oil-immersion objective (Olympus), 100 frames were acquired every 0.5 min for a total of 20 min, with a pixel size of 117 nm. Experimental procedures were approved by the Bioethics Committee of the Biotechnology Institute of the National Autonomous University of Mexico.