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 MgSO4•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.