Figure 1
Involvement of G-protein βγ and TRPCs in human sperm thermotaxis.
(a) Illustration of the thermoseparation tube. Compartment #1 was filled with spermatozoa in capacitating medium (70 × 106 cells/ml) and the other two—with the medium only. (b) Time-dependent sperm accumulation in compartment #3 when the tube is in a linear temperature gradient (34–39 °C) or in a constant, uniform temperature (no-gradient control at 34 °C or 39 °C). Each result is the mean ± SEM of 3 determinations. (c) Comparison between sperm accumulation (mean ± SEM; n = 12) in the warmest compartments of two- and three-compartment tubes following the separation period (15 and 20 min, respectively). The accumulations comprised 7.5 ± 1.0% and 4.4 ± 0.7% of the total number of spermatozoa with and without the gradient in the two-compartment tube, respectively and 1.8 ± 0.2% and 0.013 ± 0.001% in the three-compartment tube. *P < 0.01 according to paired two-tailed Student’s t-test (for the difference between the gradient and no-gradient control in the two-compartment tube), Wicoxon Signed Ranks test (for this difference in the three-compartment tube) and unpaired two-tailed Student’s t-test (for the difference between these two differences). (d) Effects of M119K on the relative sperm accumulation, VSL and percentage of motile cells (MOT). The absolute value of sperm accumulation in the warmer compartment (#3) in the absence of M119K (defined as 1) was (122 ± 11)×104 cells/ml (mean ± SEM of 9–18 determinations). *P < 0.02, **P ≤ 0.002 according to two-way ANOVA. The slope of the relative-accumulation curve was significantly different from the other two slopes (P < 0.001 according to three-way ANOVA). The absolute value of the no-gradient accumulation in compartment #3 was (2.0 ± 0.3)×104 cells/ml independently of whether M119K was present or not. (e) Effects of Pyr3 (1 μM), SKF96365 (1 μM) and NNC (10 μM) on sperm thermotaxis, as reflected in the relative accumulation in the warmer compartment. The absolute values of sperm accumulation in the absence of the inhibitors were (144 ± 27)×104, (146 ± 11)×104 and (149 ± 26)×104 cells/ml for Pyr3, SKF96365 and NNC (mean ± SEM of 9, 13 and 18 determinations, respectively). *P = 0.02, **P = 0.001 according to two-way ANOVA. The no-gradient accumulation was (1–4)×104 cells/ml independently of whether the inhibitors were present or not. (f) Inhibition of CatSper by NNC. A representative plot of a population assessment showing Ca2 + intracellular levels in human spermatozoa as fluorescence of the Ca2 + -sensitive dye Fluo-3. Prior to the measurements the relevant samples were incubated for 5 min with NNC at the indicated concentrations. The progesterone concentration was 1 μM.
