Calmodulin inhibitors increase the affinity of Merocyanine 540 for boar sperm membrane under non-capacitating conditions

Abstract

We aimed to test whether the calmodulin (CaM) inhibitors, calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), can be used to assess lipid disorder by flow cytometry using Merocyanine 540 (M540). Boar spermatozoa were incubated in non-capacitating conditions for 10 min at room temperature with 1 μM CZ, 200 μM W-7, or 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP). Then, sperm were 1) directly evaluated, 2) centrifuged and washed prior to evaluation, or 3) diluted with PBS prior to evaluation. Direct evaluation showed an increase in high M540 fluorescence in spermatozoa treated with the inhibitors (4.7 ± 1.8 [control] vs. 70.4 ± 4.0 [CZ] and 71.4 ± 4.2 [W-7], mean % ± SD, P < 0.001); washing decreased the percentage of sperm showing high M540 fluorescence for W-7 (4.8 ± 2.2, mean % ± SD) but not for CZ (69.4 ± 3.9, mean % ± SD, P < 0.001), and dilution showed an increase in high M540 fluorescence for both CZ and W-7; 8-Br-cAMP did not induce a rise in sperm showing high M540 fluorescence. Therefore, special care must be taken when M540 is used in spermatozoa with CaM inhibitors.

Keywords: Boar spermatozoa; Calmodulin (CaM) inhibitors; Merocyanine 540 (M540).

Fig. 1.

Effect of calmidazolium (CZ) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on high M540 fluorescence. Spermatozoa were incubated in the absence or presence of the inhibitors (1 μM CZ or 200 μM W-7) or with 1 mM 8-Br-cAMP in TBM for 10 min at room temperature and analyzed by flow cytometry directly (Control), after washing to remove the inhibitors (Washing), or after dilution in PBS (Diluted). Results are expressed as the percentage of viable spermatozoa (Yopro-1 negative) showing high M540 fluorescence (mean ± SD), n = 7; * P < 0.001.

Fig. 2.

Representative dot plots for the different spermatozoa treatments using the dilution protocol (A: control; B: 1 μM CZ; C: 200 μM W-7; and D: 1 mM 8-Br-cAMP). R5: live spermatozoa (YoPro-1 negative) showing low M540 fluorescence; R6: live spermatozoa (YoPro-1 negative) showing high M540 fluorescence; R7: dead spermatozoa (YoPro-1 positive).

Fig. 3.

Effect of calmidazolium (CZ) on tyrosine phosphorylation of boar sperm proteins. Spermatozoa were incubated in the absence or presence of 1 μM CZ or 1 mM 8-Br-cAMP in TBM for 10 min at room temperature or 4 h in TCM at 38.5ºC (to induce sperm capacitation). After incubation, spermatozoa were centrifuged, lysed, and the sperm proteins were analyzed by western blotting. The immunoblot shown (upper panel) is a representative of five replicates (n = 5); the p32 protein (arrow) is phosphorylated after incubation in TCM (positive control). As a loading control, an antibody against α-tubulin was included (lower panel).