All chemicals were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) unless indicated otherwise.

Semen samples

Seminal samples (N = 10, each coming from a different boar) were purchased from a local farm (Gepork S.A.; Les Masies de Roda, Spain), operating under standard commercial conditions. Animals were sexually mature (between 18 and 24 months of age), from the Piétrain breed, and were lodged under standard conditions of temperature and humidity, fed a standard diet, and provided with water ad libitum. Handling of boars by the farm staff followed the guidelines for animal welfare established by the Animal Welfare Regulations issued from the Regional Government of Catalonia (Barcelona, Spain). As authors did not manipulate any animal and the seminal doses involved in the study were originally intended to artificial insemination, no specific approval from an ethics committee was needed.

Animals were collected through the gloved-hand technique. Briefly, the sperm-rich fraction of each ejaculate was immediately filtered through a gauze to remove the gel, and diluted 1:1 (v: v) in a long-term extender (Vitasem, Magapor S.L., Zaragoza, Spain) at 37 °C inside a collecting recipient. Commercial doses were obtained after further dilution and packaging into 90-mL bags at a concentration of 3 × 109 sperm/dose. Seminal doses were then cooled to 17 °C, and three doses per ejaculate/animal were sent to our laboratory in a heat-insulating container at 17 °C. Once in the laboratory, sperm quality was assessed to ensure that all seminal doses fulfilled the minimum quality standards (viable sperm ≥ 80%; total motile sperm ≥ 70%; and morphologically normal sperm ≥ 85% [42]).

Experimental design

The presence of NCX1, NCX2 and NCX3 in the sperm plasma membrane was determined through immunofluorescence and immunoblotting. Following this, the physiological role of NCX during in vitro capacitation and acrosome reaction was analyzed by blocking the forward and reverse transport of Ca2+ via these exchangers with 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400) and ethyl 2-[[4-[(4-nitrophenyl)methoxy]phenyl]methyl]-1,3-thiazolidine-4-carboxylate (SN-6), respectively. Pharmacological blockers were added at the beginning of the experiment (0 min). Three concentrations of each blocking agent, aiming at achieving partial and complete inhibition, were tested. These concentrations, which were 0.5, 5 and 50 µM for SEA0400, and 0.3, 3 and 30 µM for SN-6, were established on the basis of preliminary experiments and the literature [28, 35, 36, 44].

For each independent experiment, the three seminal doses coming from the same ejaculate/animal were pooled and centrifuged at 600× g and 17 °C for 5 min; sperm pellets were then resuspended in capacitating medium (TCM: 20 mM HEPES, 100 mM NaCl, 3.1 mM KCl, 5 mM glucose, 21.7 mM sodium L-lactate, 1 mM sodium pyruvate, 0.3 mM Na2HPO4, 0.4 mM MgSO4·7 H2O, 4.5 mM CaCl2·2 H2O, 5 mg/mL bovine serum albumin (BSA), and 15 mM sodium bicarbonate) to a final concentration of 1 × 107 sperm/mL. Aliquots were distributed into control samples (without blocker) and blocked samples (with either SEA0400 or SN-6 at the aforementioned concentrations). Samples were incubated at 38.5 °C, 100% humidity and 5% CO2 (Binder GmbH, Tuttlingen, Germany) for 180 min; in all samples, 10 µg/mL progesterone was added at 120 min. Analysis of sperm variables was conducted after 0, 60, 120, 130, and 180 min of incubation.

At each relevant time point, sperm motility and kinematics were assessed with a computer-assisted sperm analysis (CASA) system, whereas membrane lipid disorder, acrosome integrity, mitochondrial membrane potential, tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides were determined by flow cytometry.

To confirm that SEA0400 blocks the forward transport and SN-6 blocks the reverse transport of NCX channels, the same set of experiments was conducted in sperm samples incubated under non-capacitating conditions (Tris Buffer Medium, TBM: 20 mM HEPES, 112 100 mM NaCl, 4.7 mM KCl, 5 mM glucose, 21.7 mM sodium L-lactate, 1 mM sodium pyruvate, 0.3 mM Na2HPO4, and 0.4 mM MgSO4·7 H2O). Each individual experiment included a positive control (sperm incubated in TCM without any blocker), a negative control (sperm incubated in TBM without any blocker) and blocked samples (sperm incubated in TBM with either 50 µM SEA0400 or 0.3, 3 or 30 µM SN-6). Sperm variables were analyzed at 0, 60, 120, 130, and 180 min of incubation, as previously indicated. Results are shown in Supplementary Figs. 4–14.

Immunoblotting

Immunoblotting assays were conducted following a previously described protocol [58]. Briefly, for total protein extraction, sperm pellets were resuspended in RIPA lysis buffer (R0278), supplemented 1:100 (v: v) with a commercial protease inhibitor cocktail (P8340) containing 0.1 mM phenyl-methane-sulfonylfluoride (PMSF) and 700 mM sodium orthovanadate. Samples were then incubated in agitation at 4 °C for 30 min, sonicated on ice three times (five pulses each; 20 kHz) every 2 min, and centrifuged at 10,000× g and 4 °C for 15 min to collect supernatants. Quantification of total protein in supernatants was carried out in triplicate using a detergent compatible (DC) method (BioRad, Hercules, CA, USA). Once quantified, samples were diluted to 1 µg total protein/µL in lysis buffer; 10 µL of each sample were mixed with 10 µL of 4× Laemmli reducing buffer containing 5% β-mercaptoethanol (BioRad). Samples were incubated at 95 °C for 5 min and loaded onto 12% polyacrylamide gels (Mini-PROTEAN® TGX™ Precast Gels, BioRad). Gels were run at 20 mA and 120–150 V through an electrophoretic system (IEF Cell Protean System, BioRad). Proteins from gels were then transferred onto polyvinylidene fluoride membranes using a Trans-Blot® Turbo™ device (BioRad). Thereafter, protein bands were visualized under UV exposition and scanned using a G: BOX Chemi XL system (SynGene, Frederick, MD, USA). Membranes were blocked with 1× TBS containing 10 mM Tris (Panreac, Barcelona, Spain), 150 mM NaCl (LabKem, Barcelona, Spain), 0.05% (w: v) Tween-20 (pH adjusted to 7.3; Panreac, Barcelona, Spain), and 5% bovine serum albumin (BSA, Roche Diagnostics, S.L.; Basel, Switzerland) at room temperature and agitation for 1 h.

Membranes were subsequently incubated with specific primary antibodies against NCX1 (SLC8A1), NCX2 (SLC8A2), or NCX3 (SLC8A3) (Alomone Labs, Jerusalem, Israel), which were previously diluted in blocking solution at 1:2,000 (v: v), at 4 °C overnight under agitation. Membranes were then rinsed three times with washing solution (1× TBS-Tween20), and incubated at room temperature under agitation for 1 h with an anti-rabbit secondary antibody conjugated with horseradish peroxidase (ref. P0448, Agilent, Santa Clara, CA, USA) diluted at 1:5,000 (v: v) in blocking solution. Membranes were rinsed five times with washing solution and protein bands were visualized with a chemiluminescent substrate (Immobilion™ Western Detection Reagents; Millipore, Darmstadt, Germany) and scanned with G: BOX Chemi XL 1.4 (SynGene, Cambridge, UK). The specificity of primary antibodies was confirmed through peptide competition assays using a specific blocking peptide for each primary antibody (Alomone Labs) at a concentration five times higher than the primary antibody.

Immunofluorescence

Sperm samples were washed with PBS (pH = 7.3) at 500× g and room temperature for 5 min, and then fixed with 4% (w: v) paraformaldehyde at room temperature for 30 min. After fixation, samples were washed twice with PBS at 500× g and room temperature for 5 min, and resuspended in PBS (final concentration: 5 × 106 sperm/mL). Next, 150 µL of each sperm sample was placed onto ethanol-rinsed slides and incubated at room temperature for 1 h to promote cell adhesion. Adhered sperm cells were permeabilized by incubation with 1% Triton X-100 in PBS. Next, antigens were unmasked according to the protocol of Kashir et al. [59]. In brief, slides were exposed to acidic Tyrode’s solution for 20 s, and the acid was then neutralized by washing three times with neutralization solution (Tris 100 mM, pH = 8.5), and three times with PBS.

To block nonspecific binding sites, samples were incubated with a blocking solution consisting of 5% BSA in PBS at room temperature for 1 h. Subsequently, sperm were incubated with primary NCX antibodies, either NCX1, NCX2 or NCX3 (Alomone Labs), diluted 1:100 (v: v) at room temperature for 1 h. After washing five times with PBS (5 min per wash), samples were incubated with a secondary anti-rabbit antibody Alexa Fluor™ Plus 488 (ref. A32731, Invitrogen, Waltham, MA, USA) diluted 1:200 (v: v) in blocking solution at room temperature for 1 h. Samples were again washed five times with PBS (5 min per wash), air dried and mounted with 10 µL of ProLongTM Glass Antifade Mountant with NucBlue™ (Hoechst 33342; ref. P36985, Invitrogen) in the dark. Specificities of primary antibodies were confirmed through peptide competition assays using a specific blocking peptide for each primary antibody; in all cases, the blocking peptide was five times in excess with regard to the primary antibody.

Sperm were examined under a confocal microscope (CLSM Nikon A1R; Nikon Corp, Tokyo, Japan). Samples were excited at 405 nm to localize the Hoechst 33,342-stained nuclei, and then at 488 nm to determine the localization of NCX1, NCX2, and NCX3.

Evaluation of sperm motility

Sperm motility was evaluated using a CASA system, which consisted of a phase contrast microscope (Olympus BX41; Olympus, Tokyo, Japan) equipped with a warmed stage, a video camera and the ISAS software (Integrated Sperm Analysis System V1.0; Proiser SL, Valencia, Spain). Three µL of each sample was placed into a prewarmed (38 °C) Leja chamber (IMV Technologies, L’Aigle, France) and observed under a negative phase-contrast field (Olympus 10 × 0.30 PLAN objective). At least 1,000 sperm were examined per replicate, and three replicates per sample were evaluated.

For each sperm sample and concentration of inhibitor, percentages of total and progressively motile sperm were determined. Furthermore, different sperm kinematic parameters, including curvilinear velocity (VCL, µm/s); straight line velocity (VSL, µm/s); average path velocity (VAP, µm/s); amplitude of lateral of head displacement (ALH, µm); beat cross frequency (BCF, Hz); linearity (LIN, %), which was calculated assuming that LIN = VSL/VCL × 100; straightness (STR, %), resulting from VSL/VAP × 100; and motility parameter wobble (WOB, %), obtained from VAP/VCL × 100, were measured. A sperm cell was classified as motile when its VAP was equal to or greater than 10 μm/s and progressively motile when its STR was equal to or greater than 45%. For each treatment and incubation time, motility parameters were expressed as the mean ± standard error of the mean (SEM; n = 10).

Flow cytometry

Flow cytometry was used to determine membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), tyrosine phosphorylation of sperm proteins, and intracellular levels of Ca2+, reactive oxygen species (ROS) and superoxides. Each sperm parameter was evaluated with a proper combination of fluorochromes, all being purchased from ThermoFisher Scientific (Waltham, MA, USA). Before staining, all samples were diluted to a final concentration of 1 × 106 sperm/mL and incubated at 38 °C in the dark after the addition of the corresponding fluorochromes. For each parameter, a total of three replicates per sample were examined.

Samples were evaluated using a CytoFLEX cytometer (Beckman Coulter; Fullerton, CA, USA). All samples were excited with a blue laser (488 nm). The FITC filter (525/40) was used for YO-PRO-1, PNA-FITC, Fluo4, JC-1 monomers (JC-1mon) and 2’,7’-dichlorofluorescein (DCF) fluorochromes. The PE filter (585/42) was utilized to detect ethidium (E), JC-1 aggregates (JC-1agg) and merocyanine 540 (M540) fluorochromes. APC (660/20) and PC5.5 (690/50) filters were employed for AlexaFluor647-conjugated anti-pTyr antibody and propidium iodide (PI), respectively. Flow rate and gain were not altered throughout the experiment.

Membrane lipid disorder

Lipid disorder of sperm plasma membrane was evaluated following the protocol of Rathi et al. [60], as modified by Yeste et al. [61]. Sperm were incubated with M540 (10 nM) and YO-PRO-1 (31.25 nM) at 38 °C for 10 min. M540 is a hydrophobic fluorochrome that can intercalate within the membrane. As membrane fluidity increases M540 uptake, this fluorochrome is considered as a reliable marker for destabilization of sperm plasma membrane, and has been validated in many species, including the porcine [62]. YO-PRO-1 is a vital stain that only labels sperm with an increased membrane permeability. Four sperm populations were identified: (1) viable sperm with low membrane lipid disorder (M540−/YO-PRO-1−), (2) viable sperm with high membrane lipid disorder (M540+/YO-PRO-1−), (3) non-viable sperm with low membrane lipid disorder (M540−/YO-PRO-1+), and (4) non-viable sperm with high membrane lipid disorder (M540+/YO-PRO-1+). Results are expressed as the percentage viable sperm with low (M540−/YO-PRO-1−) and high (M540+/YO-PRO-1−) membrane lipid disorder (mean ± SEM; n = 10).

Acrosome integrity

Acrosome integrity was evaluated following the modified protocol of Cooper and Yeung [63]. Samples were incubated with LIVE/DEAD working solution (Thermo Fisher Scientific, Massachusetts, USA) at 38 °C for 20 min in the dark, then centrifuged at 1,000× g and room temperature for 3 min. Samples were subsequently resuspended in blocking solution (PBS + 4 mg/mL bovine serum albumin, BSA) and centrifuged again at 1,000× g for 3 min. Pellets were resuspended in ice-cold methanol for 30 s, centrifuged at 1,000× g for 3 min, and resuspended again in 250 µL PBS; PNA-FITC (final concentration: 1.17 µM) was immediately added to resuspended samples, which were then incubated at 38 °C in the dark for 15 min. After incubation, samples were centrifuged at 1,000× g for 3 min and pellets were resuspended in 150 µL PBS. Four sperm populations were identified in dot-plots: (1) viable sperm with an intact acrosome (PNA-FITC+/PI−), (2) viable sperm with an exocytosed acrosome (PNA-FITC−/PI−), (3) non-viable sperm with an intact acrosome (PNA-FITC+/PI+), and (4) non-viable sperm with an exocytosed acrosome (PNA-FITC−/PI+). Results are expressed as the percentage of viable sperm (PI−) with either an intact (PNA-FITC+) or an exocytosed acrosome (PNA-FITC−) (mean ± SEM; n = 10).

Mitochondrial membrane potential (MMP)

Determination of mitochondrial membrane potential (MMP) was performed through staining with JC-1 (final concentration: 750 nM), diluted at 1:8,000 (v: v) in PBS, and fixable far-red LIVE/DEAD [64]. After staining, samples were incubated at 38 °C in the dark for 30 min. High MMP results in JC-1 aggregation (JC-1agg) and the subsequent emission of red fluorescence; in contrast, in sperm cells with low MMP, JC-1 remains as a monomer (JC-1mon) and emits green fluorescence (JC-1−). Four populations were distinguished: (1) viable sperm with low MMP (JC1mon/PI−), (2) viable sperm with high MMP (JC-1agg/PI−), (3) non-viable sperm with low MMP (JC-1mon/PI+), and (4) non-viable sperm with high MMP (JC1agg/PI+). For each treatment and incubation time, results are expressed as percentages of viable sperm with low (JC-1mon/PI−) and high (JC-1agg/PI−) MMP (mean ± SEM; n = 10).

Intracellular levels of Ca2+

Intracellular levels of Ca2+ were evaluated through double staining with Fluo4-AM and PI. Fluo4-AM is able to penetrate sperm cells, bind Ca2+ and emit green fluorescence. Sperm were incubated with Fluo4-AM (final concentration: 1.17 µM) and PI (final concentration: 5.6 µM) at 38 °C for 10 min. Four sperm populations were identified in the dot-plots: (1) viable sperm with low Ca2+ levels (Fluo4−/PI−), (2) viable sperm with high Ca2+ levels (Fluo4+/PI−), (3) non-viable sperm with low Ca2+ levels (Fluo4−/PI+), and (4) non-viable sperm with high Ca2+ levels (Fluo4+/PI+). Data are shown as percentages of viable sperm with high Ca2+ levels (Fluo4+/PI−), and the geometric mean intensity of Fluo4 in the Fluo4+/PI− population (mean ± SEM; n = 10).

Intracellular levels of reactive oxygen species (ROS)

Intracellular levels of total ROS were determined through staining with 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA) and PI, following the protocol of Guthrie and Welch [65] with minor modifications. Briefly, sperm were incubated with H2DCFDA (final concentration: 350 nM) at 38 °C for 20 min in the dark, and then with PI (final concentration: 6 µM) at the same conditions for further 5 min. H2DCFDA is a non-fluorescent agent that can enter the sperm cell and react with ROS, thus converting into 2’,7’-dichlorofluorescein (DCF+), a green-fluorescent molecule. The following four populations were distinguished in dot plots: (1) viable sperm with low ROS levels (DCF−/PI−), (2) viable sperm with high ROS levels (DCF+/PI−), (3) non-viable sperm with low ROS levels (DCF−/PI+), and (4) non-viable sperm with high ROS levels (DCF+/PI+). Results are expressed as percentages of viable sperm with low (DCF−/PI−) and high ROS levels (DCF+/PI−), and the geometric mean of DCF+-fluorescence intensity in the DCF+/PI− population (mean ± SEM; n = 10).

Intracellular levels of superoxides

Intracellular levels of superoxides (O2•−) were evaluated through double-staining with hydroethidine (HE) and YO-PRO-1 [65]. Sperm were incubated with 5 µM HE and 25 nM YO-PRO-1 at 38 °C in the dark for 20 min. HE permeates the sperm plasma membrane and is oxidized into ethidium (E+), which emits red fluorescence, by O2•−. Again, four separate populations were identified in dot-plots: (1) viable sperm with low superoxide levels (E−/YO-PRO-1−), (2) viable sperm with high superoxide levels (E+/YO-PRO-1−), (3) non-viable sperm with low superoxide levels (E−/YO-PRO-1+), and (4) non-viable sperm with high superoxide levels (E+/YO-PRO-1+). Results are expressed as percentages of viable sperm with low (E−/YO-PRO-1−) and high superoxide levels (E+/YO-PRO-1−), and the geometric mean of E+-fluorescence intensity in the E+/YO-PRO-1− population (mean ± SEM; n = 10).

Tyrosine-phosphorylation of sperm proteins

Analysis of tyrosine-phosphorylation (pTyr) of sperm proteins was conducted as described in Peris-Frau et al. [66]. Three separate tubes were prepared. Sperm were first stained with the far-red LIVE/DEAD fluorochrome (ThermoFisher Scientific) at 38 °C in the dark for 20 min. Samples were subsequently centrifuged at 1,000× g and room temperature for 3 min, resuspended in 10 mL blocking buffer (5% BSA in PBS), incubated for 1 min and centrifuged again at the same conditions. Then, sperm pellets were resuspended in 4% paraformaldehyde and incubated at room temperature for 15 min. Samples were then centrifuged at 1,000× g and room temperature for 3 min, resuspended in PBS and stored at 4 °C overnight. Thereafter, samples were centrifuged at 1,000× g and room temperature for 3 min, and resuspended in permeabilization buffer (0.5 g BSA, 100 µL Triton X-100, and 0.02 g sodium azide in 10 mL PBS) and incubated at room temperature for 60 min. After centrifugation at 1,000× g and room temperature for 3 min, sperm pellets from two of the three tubes were resuspended with the antibody solution (blocking solution with anti-pTyr antibody conjugated with AlexaFluor647 (Abcam) at 1:1,000). The third tube was incubated with blocking solution in the absence of the antibody, and all tubes were incubated together, at 4 °C overnight and agitation in the dark. Samples were centrifuged at 1,000× g and room temperature for 3 min, resuspended in PBS and analyzed with the flow cytometer. Four populations were identified: (1) viable sperm with low pTyr levels, (2) viable sperm with high pTyr levels, (3) non-viable sperm with low pTyr levels, and (4) non-viable sperm with high pTyr levels. Results are expressed as percentages of viable sperm with high pTyr levels (pTyr+/viable sperm), and fluorescence intensity of pTyr in the pTyr+/viable sperm population (mean ± SEM; n = 10).

Statistical analyses

Data were analyzed using a statistical package (IBM SPSS Statistics 27.0; Armonk, New York, NY, USA). Normal distribution of data and homogeneity of variances were checked with Shapiro-Wilk and Levene tests, respectively. Following this, a linear mixed model was run with each sperm parameter being considered the independent variable. The intrasubject factor was the incubation time (0, 60, 120, 130, and 180 min), and the intersubject factor was the treatment (control and samples blocked with either SEA0400 or SN-6 at the aforementioned concentrations). The post-hoc Sidak test was used for pairwise comparisons. The level of significance was set at P ≤ 0.05 in all analyses, and data are shown as mean ± SEM.

Fig. 1

Identification of NCX isoforms. Identification of NCX1, NCX2 and NCX3 in porcine sperm. Representative immunoblots using sperm samples from different boars with A) anti-NCX1, (B) anti-NCX2 and (C) anti-NCX3 antibodies, and their respective blocking peptides

Fig. 2

Immunolocalization of NCX1 isoform. Localization of NCX1 in the plasma membrane of porcine sperm (A–C), and after the peptide competition assay (D–F). NCX1 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Fig. 3

Immunolocalization of NCX2 isoform. Localization of NCX2 in the plasma membrane of porcine sperm (A–C), and after the peptide competition assay (D–F). NCX2 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Fig. 4

Immunolocalization of NCX3 isoform. Localization of NCX3 in the plasma membrane of porcine sperm (A–C), and after the peptide competition assay (D–F). NCX3 appears stained in green (Alexa Fluor 488) and nuclei in blue (DAPI; 4′6′-diamidion-2-phenylindole). Scale bar: 15 μm

Fig. 5

Sperm motility. Percentages of total (A) and progressively (B) motile sperm during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different numeral superscripts indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 6

Sperm kinematics (I). Sperm velocity parameters of VCL (A), VSL (B), and VAP (C) during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 7

Sperm kinematics (II). Amplitude of lateral head displacement (ALH, A) and beat cross frequency (BCF, B) during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 8

Acrosome integrity. Percentages of viable sperm with an intact acrosome (PNA-FITC+/EthD-1-, A) and with an exocytosed acrosome (PNA-FITC-/EthD-1-, B) during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 9

Intracellular Ca2+ levels. Percentages of viable sperm with high intracellular Ca2+ levels (Fluo4+/PI−, A) and fluorescence intensity of Fluo4+ in viable sperm (B) of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 10

Mitochondrial membrane potential. Percentages of viable sperm with low (JC-1mon/PI-, A) and high (JC-1agg/PI-, B) mitochondrial membrane potential during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 11

Total ROS levels. Percentages of viable sperm with low (DCF−/PI−, A) and high (DCF+/PI−, B) ROS levels and fluorescence intensity of DCF+ in viable sperm (C) during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 12

Superoxide levels. Percentages of viable sperm with low (E−/YO-PRO-1−, A) and high (E+/YO-PRO-1−, B) superoxide levels and fluorescence intensity of E+ in viable sperm (C) during in vitro capacitation of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)

Fig. 13

Tyrosine phosphorylation of sperm proteins. Percentages of viable sperm with phosphorylated tyrosines (pTyr+, A) and fluorescence intensity of pTyr+ in viable sperm (B) of control samples and samples blocked with either SEA0400 (0.5, 5, and 50 µM) or SN-6 (0.3, 3, and 30 µM). Different superscript letters indicate significant differences between control and blocked samples within a single time point (P < 0.05). Different superscript numbers indicate significant differences between time points within a treatment (P < 0.05). The arrow indicates the addition of 10 µg/mL of progesterone at 120 min of incubation. Results are expressed as the mean ± SEM (n = 10)