HVCN1 Channels Are Relevant for the Maintenance of Sperm Motility During In Vitro Capacitation of Pig Spermatozoa

Abstract

The objective of the present study was to determine the physiological role of voltage-gated hydrogen channels 1 (HVCN1 channels) during in vitro capacitation of pig spermatozoa. Sperm samples from 20 boars were incubated in capacitating medium for 300 minutes (min) in the presence of 2-guanidino benzimidazole (2-GBI), a specific HVCN1-channel blocker, added either at 0 min or after 240 min of incubation. Control samples were incubated in capacitating medium without the inhibitor. In all samples, acrosomal exocytosis was triggered with progesterone after 240 min of incubation. Sperm viability, sperm motility and kinematics, acrosomal exocytosis, membrane lipid disorder, intracellular calcium levels and mitochondrial membrane potential were evaluated after 0, 60, 120, 180, 240, 250, 270 and 300 min of incubation. While HVCN1-blockage resulted in altered sperm viability, sperm motility and kinematics and reduced mitochondrial membrane potential as compared to control samples, at any blocker concentration and incubation time, it had a non-significant effect on intracellular Ca²⁺ levels determined through Fluo3-staining. The effects on acrosomal exocytosis were only significant in blocked samples at 0 min, and were associated with increased membrane lipid disorder and Ca²⁺ levels of the sperm head determined through Rhod5-staining. In conclusion, HVCN1 channels play a crucial role in the modulation of sperm motility and kinematics, and in Ca²⁺ entrance to the sperm head.

Keywords: 2-GBI; HVCN1 channels; boar sperm; in vitro capacitation; progesterone.

Figure 1

Representative immunoblots for HVCN1 channel. (A) Anti-Hv1 in two pig sperm samples (S1 and S2), and its corresponding peptide competition assay; (B) Anti-Hv1 + BP. The arrow indicates the HVCN1-specific band of 70 kDa. TGX™ Stain Free (Total protein) and α-tubulin (Anti-α-tubulin) were performed as loading controls.

Figure 2

Percentages of total and progressively motile sperm throughout the incubation in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI. 2-GBI blocker was added to capacitating medium either at 0 min in experiment 1 (A,B) or at 240 min in experiment 2 (C,D; acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between treatments within the same time point, and different superscript numbers (1–5) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± standard error of the mean (SEM) (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 3

Kinematic parameters of curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) throughout the incubation of sperm in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI. 2-GBI blocker was added to capacitating medium either at 0 min in experiment 1 (A–C) or after 240 min of incubation in experiment 2 (D–F; acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between treatments within the same time point, and different superscript numbers (1–3) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 4

Indexes of linearity (LIN), straightness (STR) and oscillation (WOB) throughout the incubation of sperm in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI, added at either 0 min in experiment 1 (A–C) or 240 min in experiment 2 (D–F; acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between treatments within the same time point, and different superscript numbers (1–3) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 5

Amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) throughout the incubation of sperm in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI. The blocker was added to capacitating medium either at 0 min in experiment 1 (A–C) or after 240 min of incubation in experiment 2 (D–F; acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–3) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 6

Percentages of viable sperm (SYBR14⁺/PI^-) throughout the incubation of sperm samples in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI. The blocker was added to capacitating medium either at 0 min in experiment 1 (A) or after 240 min of incubation in experiment 2 (B, acute). Different superscript letters (a–d) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–7) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 7

Percentages of viable sperm with exocytosed acrosome (PNA⁻) in relation to total viable sperm (EthD-1⁻) throughout the incubation of sperm samples in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI. The blocker was added to capacitating medium either at 0 min in experiment 1 (A) or after 240 min of incubation in experiment 2 (B, acute). Different superscript letters (a–d) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–5) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 8

Percentages of viable sperm with high lipid disorder (M540⁺) in relation to total viable sperm (YO-PRO-1⁻) throughout the incubation of sperm samples in capacitating medium (control samples) and capacitating medium plus 1 mM, 5 mM and 10 mM 2-GBI. The inhibitor was added to capacitating medium either at 0 min in experiment 1 (A) or after 240 min of incubation in experiment 2 (B, acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–5) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 9

Percentages of viable sperm with high intracellular calcium levels evaluated through Fluo3 staining (Fluo3⁺) in relation to viable sperm (PI^-), and fluorescence intensity of Fluo3⁺ in viable sperm throughout incubation in capacitating medium (control samples) or capacitation medium plus 1 mM, 5 mM or 10 mM 2-GBI blocker, added at either 0 min in experiment 1 (A,B) or 240 min in experiment 2 (C,D; acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–5) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 10

Percentages of viable sperm with high intracellular calcium levels evaluated through Rhod5 (Rhod5⁺) in relation to total viable sperm (YO-PRO-1⁻), and fluorescence intensity of Rhod5⁺ in viable sperm throughout incubation in capacitating medium (control samples) or capacitating medium plus 1 mM, 5 mM or 10 mM, added at either 0 min in experiment 1 (A,B) or 240 min in experiment 2 (C,D; acute). Different superscript letters (a–c) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–4) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).

Figure 11

Percentages of sperm with high mitochondrial membrane potential (JC1_agg), and orange (FL2) fluorescence intensity of JC1_agg in sperm with high mitochondrial membrane potential throughout incubation in capacitating medium (control samples) or capacitating medium plus 1 mM, 5 mM or 10 mM 2-GBI. 2-GBI blocker was added either at 0 min in experiment 1 (A,B) or after 240 min of incubation in experiment 2 (C,D; acute). Different superscript letters (a–d) indicate significant differences (p < 0.05) between samples within the same time point, and different superscript numbers (1–5) indicate significant differences (p < 0.05) between time points within a given treatment. Results are expressed as mean ± SEM (n = 10). The arrow indicates the time at which 10 µg/mL of progesterone was added to all samples (i.e., 240 min of incubation).