Calcium Imaging in Capacitated Spermatozoa

Ca²⁺ levels in the capacitated spermatozoa were detected according to the previous study [18 (link)]. Briefly, the capacitated spermatozoa were incubated with 5 μM Fluo 3-AM (Dojindo Laboratories, Kumamoto, Japan) and 0.06% pluronic F-127 for 30 min at 39 °C in the dark. The capacitated spermatozoa (3–5 × 10⁵ cells/mL) were placed on glass coverslips treated with polylysine at 0.01% in the recording chamber for 5 min, and then the external solution was infused to wash the supernatant. Imaging analysis of Ca²⁺ response in motile spermatozoa was monitored using a LSM 800 confocal microscope (Zeiss, Oberkochen, Germany). We used a sample frequency of 1 Hz, and recorded Ca²⁺ fluorescence intensity for 3–4 min after different treatments. All Ca²⁺ imaging experiments were carried out at 39 °C. Cells with uneven dye loading were excluded from the analysis. NPPC was used at 1 nM, and the inhibitor l-cis-Diltiazem was used at 50 μM with a pre-incubation of 15 sec. NPPC and the inhibitor were dropped into the recording chamber using pipette tips and the recordings were conducted in the continuous presence of stimuli. Ca²⁺-free experiments were conducted using Ca²⁺-free modified Tris-buffered medium (mTBM) without BSA, obtained by omitting Ca²⁺ and adding 1 mM EGTA. NNC 055-0396, 8-Br-cGMP, BAPTA/AM, and TMB-8 were used with 2 μM, 1 mM, 5 μM, and 10 μM, respectively. The highest level of Ca²⁺ fluorescence intensity in at least 50 spermatozoa was used for the presentation of Ca²⁺ level of spermatozoa in different treatment. Each experiment was repeated three times.