Figure 1.
Expression of SCN10A, the gene that encode Na v1.8, in different human tissues.
After 35 cycles of PCR amplification, the specific band corresponding to SCN10A was detected in human testis and placenta. M, molecular size standards. The figure shows a representative RT-PCR experiment, n = 3.
Figure 2.
Immunolocalization of the Nav1.8 protein in human sperm.
(A) Flow cytometry plots of spermatozoa after overnight labeling with a Na v1.8 antibody (red plot) and the negative control treated with secondary antibody alone (black plot), n = 9. (B) Immunofluorescence images of sperm cells stained with a primary antibody against Na v1.8. Bar graph represents the distribution of this voltage-gated sodium channel in different sperm regions and the percentage of sperm cells showing each specific localization, n = 9. Scale bar, 10 μM. (C) Western blot analysis of Nav1.8 in human sperm homogenates using the Nav1.8 rabbit polyclonal antibody ab-66743. Molecular mass is indicated on the right side of the panel. The figure is representative of results obtained in 6 separate protein preparations from 6 different donors.
Figure 3.
Effects of veratridine on human sperm motility in Ca2+-containing and Ca2+-free mHTF solution.
(A) Effects of veratridine (10 μM) on progressive motility (grade A+B sperm), non-progressive motility (grade C sperm) and immotility (grade D sperm) at different times of incubation in Ca2+-containing mHTF solution. Bars are means with SEM of 36 different experiments and represent percentage changes in motility in samples treated with veratridine relative to the value observed at the same time in solvent-treated paired controls. *P<0.05, significant difference vs. control responses. (B) Time-dependent inactivation of sperm motility after incubation in a Ca2+-free mHTF solution. Afer capacitation for 2 h at 37°C in 5% CO2, the sperm suspension was divided in two aliquots, one of them incubated in Ca2+-containing solution and the other one in Ca2+-free solution. Data points are means with SEM of 7 different experiments and represent % motile sperm (grade A+B sperm). (C) Effects of veratridine (10 μM) at different times of incubation in Ca2+-containing and Ca2+-free mHTF solution. Bars are means with SEM of 7 different experiments and represent percentage changes in progressive motility (grade A+B sperm) relative to the value observed at the same time in solvent-treated paired controls.
Figure 4.
Effects of veratridine on human sperm motility in the presence of tetrodotoxin, A-803467 or ab-66743.
The effects of veratridine (10 μM) after different incubation times were analyzed in the presence of (A) the VGSC inhibitor tetrodotoxin (TTX) (10 nM) (B) TTX (10 μM), (C) the selective Na v1.8 antagonist A-803467 (10 μM), (D) the Na v1.8 antibody ab-66743 (dilution 1:50) or the corresponding solvent. Bars are means with SEM of 6-8 different experiments and represent percentage changes in progressive motility (grade A+B sperm) relative to the value observed at the same time in the respective solvent-treated paired controls. *P<0.05, significant difference vs. responses to veratridine in the presence of the antagonist or antibody solvent.
Figure 5.
Time-dependent effects of veratridine on sperm hyperactivation and acrosome reaction (AR).
(A) Effect of veratridine (10 μM) and its solvent on hyperactivated sperm motility. Capacitated sperm were treated with veratridine or its solvent for different times and hyperactivation evaluated by computer-assisted sperm analysis (CASA). Bars are means with SEM of 7 different experiments and represent % hyperactivated sperm. (B) Effect of veratridine and A-23187 on AR. Capacitated sperm were treated with veratridine (10 μM) or A-23187 (10 μM) for different times and the acrosomal status was assessed by staining sperm with FITC-PSA. Bars are means with SEM of 5 different experiments and were calculated as: (%AR reacted spermatozoa in veratridine- or A23187-treated aliquots) – (%AR reacted spermatozoa in the corresponding solvent-treated aliquots at the same time). *P<0.05, significant difference vs. values at time =0.
Figure 6.
Effects of veratridine on intracellular free Ca2+ ([Ca2+]i) and intracellular free Na+ ([Na+]i) levels in human sperm cells.
(A,B) For [Ca2+]i measurement, cells were loaded with Fura-2 and responses to veratridine (10 μM) were determined in the presence of (A) the Na v1.8 antibody ab-66743 (dilution 1:50) (red line) or its solvent (black line) or (B) in the presence of the Na v1.8 antagonist A-803467 (1 μM) (red line) or its solvent (black line). The X axis shows time in seconds and the Y axis shows [Ca2+]i data expressed by the F340/F380 ratio. Traces are representative of typical results obtained in 5-7 different experiments for each blocker. (C) For [Na+]i measurement, cells were loaded with SBFI and responses to veratridine (10 μM) were determined in the presence of A-803467 (1 μM) (red line) or its solvent (black line). The X axis shows time in seconds and the Y axis shows [Na+]i data expressed by the F340/F385 ratio. Traces are representative of typical results obtained in 6 different experiments.