Calcium imaging

The analysis of [Ca²⁺]_i in spermatozoa or mature sperm from WT and age-matched MTMR14^-/^- mice was performed as previously described [34]. Briefly, the spermatozoa or mature sperm were isolated as described above and washed twice with HS buffer ((mM): 30 HEPES, 135 NaCl, 5 KCl, 2 CaCl₂, 1 MgSO4, 10 glucose, 10 lactic acid, and 1 pyruvic acid, pH 7.4). Cells were resuspended in 1 mL of HS and incubated with 20 μM Fura-2AM for 30 min. After two washes with HS, the adherent cells were resuspended in HS and incubated for 30 min to allow for de-esterification of the dye. Fluorimetric determination of calcium concentration was performed using a Polychrome V monochromator (Till-Photonics, Gräfelfing, Germany) and/or a charge-coupled device camera coupled to an inverted microscope (IX71, Olympus, Germany). Intracellular calcium concentrations were determined as the proportions of detected fluorescence intensities at 340 nm to 380 nm. Fluorescence images were acquired and analysed with the Metafluor for Olympus software.