Chemicals and reagents

Alexa Fluor 488-conjugated goat anti-mouse antibody, Hoechst 33342 and 2',7'-Bis-(2-Carboxyethyl)-5-(and-6)-Carboxyfluorescein-acetoxymethyl (BCECF-acetoxymethyl) (AM) ester were obtained from Molecular Probes (Ghent, Belgium). Monoclonal 4G10 Platinum anti-phosphotyrosine mouse antibodies were purchased from Millipore (Overijse, Belgium). Fatty acid-free bovine serum albumin (BSA) (A9418; cell culture tested), Triton X-100, and all other chemicals not otherwise listed were obtained from Sigma-Aldrich (Bornem, Belgium).

Semen collection and preparation

Stallion semen was collected from three adult stallions of proven fertility using a Colorado model artificial vagina (Animal Reproduction Systems; Chino, CA, USA) at the equine reproduction clinics at Ghent and Utrecht Universities. The raw ejaculate was filtered through gauze to remove the gel fraction and any debris, before visual evaluation of sperm motility by light microscopy (200×) on a heated stage at 37 °C; if the motility was acceptable (>65% motile), the semen was immediately transported to the laboratory for further processing. One milliliter of fresh semen with a concentration of 100–300 × 10⁶ spermatozoa/ml was separated through a 45/90% Percoll gradient [29, 30]. Next, the sperm pellet was washed once with noncapacitating medium (NCM) (100 mM NaCl, 4.7 mM KCl, 1.2 mM MgCl₂, 5.5 mM glucose, 22 mM 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES), 2.4 mM sodium lactate and 1.0 mM pyruvic acid; pH = 7.4 and 280–300 mOsm; [31]). Per experiment three replicates were performed using one ejaculate from three different stallions (n = 3).

Ejaculates from three boars with proven fertility were collected at AIM Varkens KI Nederland (Vaassen, The Netherlands; a commercial enterprise producing semen doses for commercial pig artificial insemination). Freshly ejaculated sperm was filtered through gauze to remove the gelatinous material and then diluted, washed, and stored in Beltsville Thawing Solution, as described previously [32]. All buffers and other solutions used were isoosmotic (285–300 mOsm) and kept at room temperature unless stated otherwise. Upon arrival in the laboratory, boar semen was processed in a similar way to stallion semen, with the exception that 1 ml semen was separated through a different density gradient, i.e., 35/70% Percoll [33].

Animal experiments

The study was approved by the Ethics Committee of the Faculty of Veterinary Medicine of Ghent University (EC2013/175 and EC2013/176). The Institutional Animal care and Use Committee of Utrecht University approved with this study.

Sperm capacitation/hyperactivation

In order to provide conditions supportive of sperm capacitation, NCM was modified by replacing sodium lactate with 2.4 mM calcium lactate and adding 25 mM NaHCO₃ and 7 mg/ml BSA (pH = 7.4; 280–300 mOsm/kg; osmolality was adjusted by graduated reduction of the NaCl to compensate for the added 25 mM NaHCO₃); this medium was pre-equilibrated for at least 2 h in a humidified atmosphere containing 5% CO₂ at 38.5 °C and is further referred to as CM (adapted from McPartlin et al. [31]). The washed sperm pellet was diluted to a concentration of 10 × 10⁶ spermatozoa/ml with CM. Hyperactivated motility was induced by resuspending the spermatozoa in (1) CM supplemented with 2.5 mM procaine hydrochloride (Sigma-Aldrich, Bornem, Belgium) at a final concentration of 1 × 10⁶ spermatozoa/ml [8]. To prepare concentrations of up to 5 mM procaine, a stock of 50 mM procaine hydrochloride dissolved in CM was pre-equilibrated for at least 2 h in a humidified atmosphere containing 5% CO₂ at 38.5 °C (to restore the pH to the physiological range; 7.2–7.4). Subsequently, 2.5 and 5 mM procaine-containing CM were prepared by diluting the stock solution with the required amount of equilibrated CM. The pH of all incubation media was adjusted to 7.2–7.4. Procaine concentrations higher than 5 mM (10, 25 or 50 mM) were made without stock preparation. To obtain medium osmolality between 280–300 mOsm/kg in these high procaine concentration capacitating media, NaCl concentration was reduced accordingly. The latter media were subsequently pre-equilibrated overnight in a humidified atmosphere containing 5% CO₂ at 38.5 °C to restore the pH to 7.2–7.4.

To investigate the effect of the Ca²⁺ in CM on hyperactived motility, calcium lactate was replaced by sodium lactate, and 2 mM of the Ca²⁺ chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) was added.

CM without NaCl was prepared by replacing 100 mM NaCl with 200 mM D-mannitol while Na⁺-free CM was made by replacing the 100 mM NaCl with 200 mM D-mannitol and 25 mM NaHCO₃ with KHCO₃. CM plus NH₄Cl was prepared by replacing the 25 mM NaCl with 25 mM NH₄Cl. CM at pH 7.9 was prepared by increasing NaHCO₃ to 100 mM and reducing NaCl accordingly, to maintain the osmolality between 280–300 mOsm/kg. All media were pre-equilibrated for at least 2 h in a humidified atmosphere containing 5% CO₂ at 38.5 °C before the start of incubation.

Oocyte maturation

Ovaries were collected from slaughtered mares (Euro Meat Group, Moeskroen, Belgium). Within 4 h after slaughter, all follicles larger than 5 mm were aspirated using a 16 gauge needle attached to a vacuum pump (∼100 mm Hg), scraped with the bevel of the aspirating needle, and flushed with Dulbecco's phosphate buffered saline (DPBS) containing 25 IU/ml heparin. A maximum of 30 cumulus-oocyte complexes (COCs) were transferred to 500 µl Dulbecco's Modified Eagle's Medium Nutrient Mixture F-12 (DMEM-F12)-based maturation medium [34] and placed in an incubator at 38.2 °C in a humidified atmosphere of 5% CO₂-in-air for 28 h. After maturation, COCs were partially or completely denuded by gentle pipetting in 0.05% bovine hyaluronidase diluted in HEPES buffered DMEM-F12 medium. Degenerated oocytes were excluded from subsequent experiments, whereas all nondegenerated oocytes were used for gamete co-incubation with the assumption that an extruded polar body was present (this could not be confirmed in partially cumulus-enclosed oocytes).

In vitro fertilization/gamete co-incubation

Equine gamete co-incubation was performed with some minor modifications in the various capacitating media described above [31]. As previously indicated, sperm was suspended at 1 × 10⁶ spermatozoa/ml in each specific CM (medium pH = 7.2–7.4; previously adjusted by incubation in an atmosphere containing 5% CO₂). About 100 µl droplets of these sperm suspensions were pipetted into Petri dishes and covered with 5% CO₂-equilibrated mineral oil. Five partially denuded mature oocytes were then transferred to each medium droplet, and the Petri dishes were incubated at 38.2 °C in 5% CO₂ in humidified air. After 18 h of co-incubation, partially denuded oocytes were fully denuded by gentle pipetting in 0.05% bovine hyaluronidase in HEPES buffered DMEM/F12. Subsequently, oocytes were checked for sperm penetration or cultured for an additional 6 h to assess oocyte nuclear configuration and second polar body formation or for 2.5 days in groups of five oocytes per 5 µl droplet of DMEM-F12 with 10% fetal calf serum, at 38.5 °C in a humidified atmosphere of 5% CO₂, 5% O₂, and 90% N₂. After IVF, oocytes were fixed at different developmental stages (zygote, 2-cell, 4–8 cell, 8–16 cell) to assess nuclear configuration, second polar body formation, and developmental stage. To control for direct effects of procaine, oocytes were incubated in similar media in the absence of sperm.

Deoxyribonucleic acid configuration and embryonic development

Oocytes and presumptive zygotes or early embryos at various developmental stages were fixed in 4% paraformaldehyde in DPBS at room temperature for 1 h. The fixative was then removed by washing the oocytes twice in wash medium (DPBS containing 0.5% BSA). Next, the fixed oocytes were incubated in a 3.2 µM Hoechst 33342 solution in wash medium for 10 min at room temperature. The oocytes were then washed four times in wash medium and mounted on siliconized glass slides (Marienfeld, Germany) using 1,4-diazabicyclo[2,2,2]octane as antifade and sealed with nail polish. Excessive pressure from the cover slip was prevented by placing a few droplets of Vaseline on the microscope slides prior to mounting. Starting from the time of incubation in Hoechst 33342, oocytes were shielded from the light to prevent fading. Mounted slides were kept at 4 °C in the dark until evaluation. Oocyte degeneration was indicated by irregular morphology of the oocyte. The Hoechst dye stained the deoxyribonucleic acid (DNA) of both the oocyte and the spermatozoa, and can thus be used for detecting possible fertilization, i.e., whether both the female and male pronuclei (PN) and the second polar body (containing condensed DNA) are all present. After 2.5 d in culture, the ability of equine oocytes/zygotes to undergo nuclear duplication and cell cleavage was assessed, with condensed DNA fragments indicative of nuclear degradation visualized using the Hoechst dye.

Sperm motility assessment

To assess the effect on sperm motility of the different capacitating strategies based respectively on procaine-pre-incubation, extracellular Ca²⁺, Na⁺ influx inhibition, or weak base activity to increase cytoplasmic pH, motility parameters for sperm in suspension were assessed using a computer-assisted sperm analyzer (CASA: Hamilton-Thorne motility analyzer Ceros version 12.3d; Hamilton-Thorne Research, Beverly, MA, USA). Under defined capacitating conditions, BSA was replaced with 0.02% polyvinyl alcohol to avoid the marked sperm agglutination noted for stallion sperm after centrifugation in BSA-containing medium [11]. For each analysis, 10 µl of sperm solution diluted in different CM was mounted on a prewarmed glass slide (Marienfeld, Lauda-Königshofen, Germany) and maintained at 37 °C using a Tokai Hit thermoplate. Five randomly selected microscopic fields in the center of the slide were scanned four times each, generating 20 scans for each sample. The mean of these five scans per sample was used for statistical analysis. The settings of the CASA-software HTR 12.3 for analyzing motility parameters of stallion sperm were based on those described by Loomis and Graham [35] and Hoogewijs et al. [36]. To evaluate the effect of Na⁺ omission, or of weak base activity to raise the cytoplasmic pH, the percentages of motile and progressively motile sperm were assessed. To assess the effect of chelating extracellular Ca²⁺, omitting Na⁺, or weak base activity on sperm hypermotility, two parameters were used, namely, amplitude of lateral head displacement (ALH, in µm; i.e., the mean width of head oscillations) and curvilinear velocity (VCL, in µm/s; i.e., the average velocity of a sperm along its actual, two-dimensional curvilinear trajectory). An increase in ALH and VCL was taken to indicate induction of hyperactivated motility [8, 10, 11].

Motility patterns of stallion sperm incubated in CM + 2.5 mM procaine, Ca²⁺-depleted CM + 2.5 mM procaine and CM + 25 mM NH₄Cl were imaged using a Moticam 10+ camera (Motic Deutschland GmbH, Wetzlar, Germany) attached to an Olympus BX40 microscope (Olympus Nederland B.V., Leiderdorp, The Netherlands) and equipped with a phase contract objective 20× (Olympus Nederland B.V., Leiderdorp, The Netherlands). Images were acquired using the Motic Images Plus 3.0 ML program (Motic Deutschland GmbH, Wetzlar, Germany).

Cytoplasmic and cell organelle pH measurements

Percoll-separated sperm was washed twice using NCM and stained with 5 µM of the cytoplasmic pH-sensitive dye BCECF-AM [10, 11] or with 5 µM of the cell organelle pH-sensitive marker Lysosensor green DND-189 [28] in NCM, by incubation at 38.5 °C for 30 min. The extracellular dye was then removed by washing the sperm twice in NCM (400 g, 5 min). The washed BCECF-AM loaded sperm (30 × 10⁶ sperm/ml) was added to NCM, CM, CM without NaCl, CM without Na⁺, CM plus 25 mM NH₄Cl, CM at pH 7.9, and CM plus 2.5, 5, 10, 25, or 50 mM procaine. Subsequently, the BCECF signal was measured in 200 µl aliquots of sperm suspension loaded into a 96-well microplate reader (CLARIOstar, BMG LABTECH, Ortenberg, Germany) and excited at 440 and 490 nm. The cytoplasmic pH was proportional to the ratio of fluorescence at 440/490 (expressed in AU). Baseline fluorescence was recorded for each well and set to ratio = 0. A similar approach was used to measure the fluorescence intensity of Lysosensor green DND-189-loaded sperm (30 × 10⁶ sperm/ml) incubated in NCM, CM, CM plus 25 mM NH₄Cl, CM at pH 7.9, and CM plus 2.5 or 10 mM procaine. Sperm samples were excited at 443 nm. Readings were taken at 0, 10, 30, and 60 min after the treatments have been added, since a rapid procaine-induced weak base activity in stallion sperm has been reported previously [11].

Cytoplasmic cAMP measurements

Since a rise in cAMP is considered to be an early capacitation response [37], stallion sperm (5 × 10⁶ sperm/ml) was incubated for 15 min and 1 h in NCM, CM, CM without NaCl, CM without Na⁺, CM plus 25 mM NH₄Cl, CM at pH 7.9, CM plus 1 mM caffeine, and CM plus 2.5, 5, 10, 25, or 50 mM procaine. Boar sperm samples incubated in NCM, CM, and CM plus 1 mM caffeine media for 15 min and 1 h were included for comparison. After 15 min and 1 h incubations, sperm samples were centrifuged for 2 min at 1200×g. The sperm pellet was resuspended in 200 µl 0.1 mM HCl, vortexed twice for 2 s and incubated for 20 min at room temperature. Subsequently, sperm samples were centrifuged at 5000×g for 5 min, and all supernatant samples were stored at –80 °C until further analysis [37]. Using a direct cAMP Enzyme Immunoassay Kit (Sigma-Aldrich, Zwijndrecht, The Netherlands), cAMP levels in acetylated sperm supernatant samples were measured by following the manufacturer's instructions. A standard curve was run for each assay (Figure 10A), and the unknown cAMP concentrations were obtained by logistic curve fitting (as recommended by the manufacturer).

Quantification of tail-associated protein tyrosine phosphorylation

Assessment of protein tyrosine phosphorylation, which is considered a hallmark of late stage capacitation, was performed as described by Leemans et al. [24]. Briefly, spermatozoa were incubated for 4 h in NCM, CM, CM without NaCl, CM without Na⁺, CM plus 25 mM NH₄Cl, CM at pH 7.9, CM plus 1 mM caffeine, and CM plus 2.5, 5, 10, 25, or 50 mM procaine media under atmospheric conditions (5% CO₂ or air). The incubated sperm suspensions were subsequently washed twice and fixed in 4% paraformaldehyde in DPBS at room temperature for 15 min. The fixative was removed by three centrifugation steps using DPBS (600 g for 5 min). The washed spermatozoa were subsequently incubated in 0.1% Triton X-100 in DPBS for 10 min at room temperature to ensure complete permeabilization of the membranes. The permeabilized spermatozoa were then incubated in blocking buffer (DPBS containing 1% BSA) for 10 min at room temperature. Next, the spermatozoa were incubated overnight at 4 °C in buffer containing 0.1% BSA and the mouse monoclonal 4G10 Platinum IgG_2b protein anti-phosphotyrosine antibody (diluted 1:500). After incubation, unbound antibody was removed by washing the spermatozoa twice with 1 mL DPBS containing 0.1% BSA (600 g for 5 min). The spermatozoa were then stained with a monoclonal goat anti-mouse antibody conjugated to Alexa Fluor 488 (Invitrogen, Molecular Probes, Ghent, Belgium) for 1 h at room temperature. After immunolabeling, the nonbound antibody conjugates were removed by washing three times with DPBS containing 0.1% BSA, and once using DPBS (600 g for 5 min). The immunolabeled spermatozoa were mounted on glass slides under a cover slip and sealed with nail polish. The proportion of spermatozoa with green fluorescent tails among the total sperm population (with Hoechst 33342 fluorescent heads) was determined by randomly scoring 200 spermatozoa. Samples were examined using a Leica DMR microscope equipped with a mercury lamp and appropriate filters, at a magnification of 400×.

Microscopic imaging

Oocyte cleavage, possible second polar body formation, and DNA configuration were examined using an inverted Nikon A1R confocal microscope (Nikon Instruments, Paris, France), mounted on a Nikon Ti body, and with a 60×/1.4 Plan Apo oil immersion lens. Hoechst was excited using a 405 nm diode laser, and a 488 nm Argon laser was used for simultaneous DIC imaging. Images were acquired using Nikon Elements Software and a pinhole setting of 1 Airy unit (AU) and constant acquisition settings (laser power, gain and offset, and scan speed). Digital optical sections were collected across an axial range that spanned the oocyte, at a step size of 1 µm.

Sperm stained for tail-associated protein tyrosine phosphorylation and a DNA counter stain were detected by means of fluorescence microscopy (400×) using a Leica DMR microscope equipped with excitation filters of 360–590 nm and a 100 W mercury lamp. Alexa Fluor 488-conjugated goat anti-mouse antibody and Hoechst 33342 were excited using 495 nm and 343 nm lasers, respectively. Emission spectra of the dyes were then filtered at 519 nm and 483 nm. These emission spectra were detected by blue (LP 425 nm) and green (LP 515 nm) filters corresponding to the emission peaks of the dyes. Images were acquired using the Image Database program (Leica, Van Hopplynus N.V., Brussel, Belgium).

Statistical analysis

All experiments were performed at least three times, and sperm cells for a given comparison were derived from independent ejaculates from each of the three Warmblood stallions. Before analysis, normality of the variables was checked using the Shapiro-Wilk and Kolmogorov-Smirnov tests (P < 0.05). The effect of different capacitating conditions, i.e., procaine-preincubation, extracellular Ca²⁺ removal, Na⁺ influx inhibition, and weak base activity, on sperm motility parameters, cytoplasmic pH, cell organelle pH, cAMP, and tail-associated protein tyrosine phosphorylation was assessed by analysis of variance (ANOVA). Overall differences were identified using repeated measures ANOVA with Greenhouse-Geisser and Bonferroni correction, as implemented in the general linear model. Scheffé post-hoc tests were performed for pairwise comparisons. Differences were considered significant if P < 0.05.

The effect of different conditions on embryo development and DNA configuration was analyzed by binary logistic regression for binomially distributed data. Where differences existed, further comparisons between groups were performed by chi-square analysis (χ² fit tests). All experiments were repeated three times. Differences were considered significant if P < 0.05. All analyses were performed using SPSS version 20 for Windows (SPSS IBM, Brussels, Belgium).

Procaine-induced hyperactivated motility is lost when procaine is washed out

After 30 min, sperm in CM supplemented with procaine demonstrated significantly higher ALH and VCL (ALH: 7.4 ± 0.1 µm and VCL: 173 ± 3 µm/s) than sperm incubated in standard CM (ALH: 3.7 ± 0.3 µm and VCL: 69 ± 1 µm/s) (P < 0.01). However, 30 min after removing procaine by washing, the ALH and VCL values of stallion sperm previously exposed to procaine had returned to preexposure levels (ALH: 3.4 ± 0.3 µm and VCL: 72 ± 2 µm/s). Similar observations were made at 2, 4, and 6 h which leads us to conclude that stallion spermatozoa only display hyperactivated motility when directly exposed to procaine (Figure 1). After 2 h incubation, ALH and VCL values decreased in all conditions (Figure 1). These data suggest that the removal of procaine before introducing the sperm to the fertilization medium to avoid the detrimental effects of procaine on equine oocytes during gamete co-incubation did not support hyperactivated sperm motility.

Procaine-induced hyperactivated motility is independent of extracellular Ca²⁺

At 0.5 h, there were no significant differences in ALH and VCL between Ca²⁺-depleted (ALH: 7.5 ± 0.3 µm and VCL: 167 ± 6 µm/s) and Ca²⁺-containing (ALH: 7.5 ± 0.4 µm and VCL: 167 ± 8 µm/s) procaine-supplemented CM, at pH = 7.4 (Figure 2). Similar observations were made at 2, 4, and 6 h incubation time points indicating that procaine-induced hyperactivated motility is independent of an extracellular Ca²⁺ source (Figure 2). After 2 h incubation, ALH and VCL values decreased in both Ca²⁺-depleted and Ca²⁺-containing, procaine-supplemented CM (Figure 2). A similar trend was observed when sperm suspensions were incubated under identical conditions at pH 7.9 (Figure 2). Video clips of stallion spermatozoa showing hyperactivated motility in 2.5 mM procaine CM and Ca²⁺-depleted 2.5 mM procaine CM + 2 mM EGTA, respectively, are attached as  Supplementary Files 1 and  2. These data suggest that hyperactivated motility in stallion sperm is independent of extracellular Ca²⁺ influx through pH-gated CATSPER channels.

Procaine-induced hyperactivated motility is not blocked by inhibiting Na⁺ influx

Percentages of motile spermatozoa did not differ significantly between four tested media (CM, CM + 2.5 mM procaine, CM lacking NaCl or all sources of Na⁺) after 5, 15, and 30 min and 1, 2, 4, or 6 h of incubation. After 2 h of incubation, percentages of motile spermatozoa decreased in all conditions tested (Figure 3A).

Significantly lower percentages of progressively motile spermatozoa were observed after 5 min incubation in procaine-supplemented CM (18 ± 4%; P < 0.01) compared to standard CM (83 ± 11%; P < 0.01), CM without NaCl (68 ± 5%; P < 0.01), and CM without Na⁺ (69 ± 12%; P < 0.01) (Figure 3B); there were no significant differences between the other three media. Similar observations were made at all other incubation time points (Figure 3B). After 2 h incubation, percentages of progressively motile spermatozoa decreased in CM without NaCl and CM without Na⁺ (Figure 3B).

ALH and VCL values indicative of hyperactivated motility showed the opposite trend. At 5 min incubation, significantly higher ALH and VCL values were observed in CM with 2.5 mM procaine (ALH: 6.9 ± 0.2 µm; VCL: 170 ± 7 µm/s) than in standard CM (ALH: 3.5 ± 0.1 µm; VCL: 71 ± 4 µm/s; P < 0.01; as previously shown [8, 10, 11]), CM without NaCl (ALH: 4.1 ± 0.2 µm; VCL: 89 ± 9 µm/s; P < 0.01), or CM without Na⁺ (ALH: 3.9 ± 0.4 µm; VCL: 90 ± 2 µm/s; P < 0.01) (Figure 3C and D). No significant differences in ALH and VCL values were observed between the latter three media. A similar pattern was observed at all other incubation time points. After 2 h incubation, ALH and VCL values decreased in CM plus 2.5 mM procaine (Figure 3C and D). These data indicate that procaine-induction of hyperactivated motility is not mediated by inhibition of Na⁺ influx.

Figure 1.

Motility patterns indicative of hyperactivated motility were assessed by CASA in stallion sperm suspensions incubated for 0.5, 2, 4, and 6 h in CM (black bars), CM + 2.5 mM procaine (grey bars), and CM after sperm being washed following pre-incubation in CM +2.5 mM procaine (white bars) (pH = 7.4) (n = 3 samples in each group; three replicates). Parameters of interest included (A) ALH and (B) VCL. Hyperactivated motility in sperm suspensions was triggered by direct exposure to 2.5 mM procaine, whereas washing to remove the procaine resulted in a complete loss of hyperactivated motility. For both ALH and VCL, values that differ significantly (P < 0.05) are indicated by different small letters.

Procaine-induced hyperactivated motility is associated with weak base activity

Percentage of motile spermatozoa (total motility) did not differ significantly between four tested media (CM, CM + 2.5 mM procaine, CM + 25 mM NH₄Cl, and CM at pH 7.9) up to 6 h. After 2 h incubation, the proportion of motile sperm had decreased in all four tested conditions (Figure 4A).

At 5 min, the percentage of progressively motile spermatozoa differed significantly between the four media tested (Figure 4B). The lowest percentage of progressively motile sperm was observed in the presence of procaine (18 ± 4%) whereas the highest was seen in standard CM (83 ± 11%). In comparison to procaine exposure, slightly but significantly higher percentages of progressively motile spermatozoa were observed for sperm suspensions incubated with NH₄Cl (31 ± 2%) while CM at pH 7.9 (43 ± 4%) yielded intermediate percentages of progressive motility (Figure 4B). Similar observations were made at all incubation time points up to 2 h. By 4 h incubation, no difference in percentage of progressively motile spermatozoa was evident between CM (55 ± 7%) and NH₄Cl-containing media (48 ± 4%) although a lower incidence of progressive motility was still evident in procaine-supplemented CM (18 ± 4%). CM at pH 7.9 (34 ± 2%) still showed intermediate percentages of progressively motile spermatozoa. Similar measurements were obtained at 6 h (Figure 4B). Parameters indicating hyperactivated motility showed the opposite trend. After 5 min incubation, peak ALH and VCL values were observed in procaine-supplemented medium (ALH: 7.1 ± 0.4 µm; VCL: 173 ± 11 µm/s) and lowest values were observed in CM (ALH: 3.6 ± 0.1 µm; VCL: 72 ± 5 µm/s). Slightly but significantly lower different ALH and VCL values than in the presence of procaine were observed for NH₄Cl-supplemented CM (ALH: 6.2 ± 0.3 µm; VCL: 160 ± 22 µm/s), and intermediate values were observed in CM at pH 7.9 (ALH: 4.6 ± 0.4 µm; VCL: 73 ± 11 µm/s) (Figure 4C and D). A similar pattern was observed through the first 2 h of incubation. At 4 h incubation, significantly higher ALH and VCL values were observed in the presence of procaine (ALH: 5.9 ± 0.7 µm; VCL: 144 ± 3 µm/s) than in the three other incubation conditions (CM: ALH: 3.3 ± 0.2 µm, VCL: 75 ± 3 µm/s; NH₄Cl supplemented CM: ALH: 3.3 ± 0.4 µm, VCL: 75 ± 7 µm/s; CM at pH 7.9: ALH: 3.5 ± 0.1 µm, VCL: 69 ± 5 µm/s) which did not differ from each other. Similar findings were apparent at 6 h (Figure 4C and D). A video clip of stallion spermatozoa demonstrating hyperactivated motility in 25 mM NH₄Cl-supplemented CM is attached as  Supplementary File 3.

Figure 2.

Motility patterns indicative of hyperactivated motility were assessed by CASA in stallion sperm incubated for 0.5, 2, 4, and 6 h in CM (black bars), CM + 2 mM EGTA (grey bars), CM + 2.5 mM procaine (white bars), and CM + 2.5 mM procaine +2 mM EGTA (striped bars) at pH = 7.4 and pH = 7.9 (n = 3 samples in each group; three replicates). Parameters of interest were (A) ALH and (B) VCL. Hyperactivated motility of spermatozoa induced by 2.5 mM procaine is independent of extracellular Ca²⁺ and pH. For both ALH and VCL, values that differ (P < 0.05) are indicated by different small letters.

Given the known effect of NH₄Cl on cytoplasmic pH [38] and hyperactivated motility [11, 38], these data suggest that procaine-induced hyperactivated motility is associated with the weak base property of procaine, which presumably elevates the cytoplasmic pH of stallion spermatozoa.

Procaine and NH₄Cl both trigger oocyte cleavage

After 2.5 days of culture, we observed a degenerative effect of 2.5 mM procaine, 25 mM NH₄Cl, and pH 7.9 on partially cumulus-denuded oocytes (PD). In capacitating conditions, 14 ± 3% PD degenerated; in 2.5 mM procaine capacitating conditions, 27 ± 3% PD degenerated; in NaCl-free conditions, 17 ± 2% PD degenerated; in Na⁺ free-conditions, 16 ± 2% PD degenerated; in NH₄Cl supplemented conditions, 35 ± 2% PD degenerated; and in pH 7.9 conditions, 52 ± 11% PD degenerated. Oocytes exposed to CM at pH 7.9 showed the highest incidence of degeneration, whereas significantly lower incidences were observed in the presence of procaine or NH₄Cl. This suggests that the intra- and extracellular pH changes induced by procaine, NH₄Cl, and pH 7.9 are toxic to equine oocytes (Figure 5).

In agreement with a previous study [10], in the presence of 2.5 mM procaine, 42 ± 3% of oocytes appeared to cleave; similarly, in the presence of NH₄Cl, 25 ± 2% of oocytes underwent apparent cleavage. No oocyte cleavage was observed in the medium from which either NaCl or all sources of Na⁺ were omitted or at pH 7.9. However, none of the oocytes that cleaved in the presence of procaine or NH₄Cl developed beyond the 8–16 cell stage and none reached the blastocyst stage. Beyond 5–6 days of incubation, any 8–16 cell stage embryos derived from either treatment began to degenerate (Figures 5 and 6). Moreover, none of these parameters differed significantly between oocytes incubated in the presence or absence of spermatozoa, indicating that procaine and NH₄Cl-induced oocyte cytokinesis was sperm-independent (P > 0.12 for all comparisons; Figure 5).

Figure 3.

Evaluation of different motility parameters ((A) percentage of total motile or TM; (B) percentage of progressively motile or PM; (C) ALH; and (D) VCL) during 6 h incubation of stallion spermatozoa in (1) CM, (2) CM + 2.5 mM procaine, (3) CM lacking NaCl, and (4) CM lacking Na⁺ (pH = 7.4) (n = 4 samples in each group; three replicates). Hyperactivated motility in sperm suspensions was triggered by direct exposure to 2.5 mM procaine, whereas the medium with reduced or no Na⁺ did not have this effect. For percentage of progressively motile, ALH and VCL, values that differ (P < 0.05) are indicated by different small letters.

Figure 4.

Evaluation of different motility parameters ((A) percentage of total motile or TM; (B) percentage of progressively motile or PM; (C) ALH; and (D) VCL) during 6 h incubation of stallion spermatozoa in (1) CM, (2) CM + 2.5 mM procaine, (3) CM + 25 mM NH₄Cl, and (4) CM pH 7.9 (n = 3 samples in each group; three replicates). Hyperactivated motility in sperm suspensions was triggered by exposure to 5 mM procaine and 25 mM NH₄Cl and to a lesser extent by CM at pH 7.9. For percentage of progressively motile, ALH and VCL, values that differ (P < 0.05) are indicated by different small letters. Repeated measure ANOVA with Greenhouse-Heisser and Bonferroni correction Scheffé post hoc tests were performed for pairwise comparison.

Figure 5.

Percentage of PD that either degenerated or cleaved after 2.5 days in culture following incubation with or without spermatozoa in CM, CM + 2.5 mM procaine, CM—NaCl, CM—Na⁺, CM + 25 mM NH₄Cl, and CM at pH 7.9. CM at pH 7.9 had a severe degenerative effect on oocytes. In the presence of 2.5 mM procaine and 25 mM NH₄Cl, oocytes cleaved up to the 8-cell stage. Data represent mean (±SD) percentages of oocytes after incubation in CM (black bars), CM + 2.5 mM procaine (dark grey bars), CM—NaCl (light grey bars), CM—Na⁺ (white bars), CM + 25 mM NH₄Cl (hatched bars), and CM pH 7.9 (dotted bars); n = 20 oocytes in each group, three replicates. Values that differ significantly (P < 0.05) are indicated by different small letters for degenerated oocytes or capitals for cleavage.

In short, exposure to procaine or NH₄Cl induced sperm-independent cleavage of horse oocytes, whereas inhibition of Na⁺ influx or exposure to a high pH medium did not.

Procaine and NH₄Cl-induced cleavage are accompanied by DNA fragmentation

After 24 h incubation, the majority of the oocytes in CM (83 ± 4%), CM without NaCl (71 ± 4%), and CM without Na⁺ (78 ± 4%) displayed a normal metaphase spindle, suggesting that inhibiting Na⁺ influx into equine oocytes does not affect DNA configuration. By contrast, the majority of oocytes exposed to procaine (75 ± 1%) or NH₄Cl (53 ± 4%) displayed DNA fragmentation (Figure 6). Similarly, equine oocytes incubated in pH 7.9 CM (89 ± 5%) also exhibited severe DNA degeneration (Figure 7).

The formation of normal-looking (pro) nuclei (1/2 pronucleus (PN)) was a rare finding in any of the conditions tested (less than 6 ± 5%; Figure 7). Moreover, completion of the second meiotic division, as evidenced by the formation of a second polar body, was not seen in any of the oocytes, suggesting that resumption of metaphase plate of meiotic division II (MII) was not initiated by any of the tested conditions.

After procaine or NH₄Cl exposure, very few of the daughter cells in apparently cleaved oocytes contained DNA at all, while others displayed condensed, fragmented pieces of DNA (Figure 7). Effects on oocyte nuclear DNA was independent of the presence or absence of spermatozoa in fertilizing medium (P > 0.09 for all comparisons; Figures 6 and 7).

In short, exposure to procaine or NH₄Cl induced DNA fragmentation in equine oocytes which underwent cytokinesis, whereas inhibition of Na⁺ influx did not (Figure 7).

Procaine elevates cytoplasmic pH in a concentration-dependent way primarily by alkalinizing acidic sperm cell organelles

The BCECF-AM ratio at 0 min in stallion sperm suspensions incubated in NCM was assigned as the baseline (3.10 ± 0.14). After 10 min incubation, no significant increase in BCECF-AM ratio was observed for sperm cells in NCM (2.95 ± 0.07), CM (3.10 ± 0.42), CM without NaCl (3.00 ± 0.28), or CM without Na⁺ (3.05 ± 0.07) (Figure 8A). In contrast, procaine induced a rapid, concentration-dependent increase in the BCECF-AM ratio during the first 10 min of incubation (after 10 min in 2.5 mM procaine: 3.75 ± 0.07; in 5 mM procaine: 4.55 ± 0.07; in 10 mM procaine: 5.60 ± 0.14; in 25 mM procaine: 11.55 ± 0.07; and in 50 mM procaine: 17.50 ± 0.00) that was sustained for the entire 60 min incubation (Figure 8A). A slight but significant increase in BCECF-AM ratio during the first 10 min was also observed for sperm cells suspended in CM plus 25 mM NH₄Cl (3.85 ± 0.07) and CM at pH 7.9 (4.10 ± 0.14) and was also maintained for the rest of the incubation (Figure 8A). Overall, it appears that weak bases, such as procaine and NH₄Cl, elevate the cytoplasmic pH in stallion sperm in a concentration-dependent fashion.

Next, baseline Lysosensor Green DND-189 fluorescence was recorded in NCM conditions at 0 min and set as 100. After 10 min incubation, no significant decrease in Lysosensor Green DND-189 fluorescence was observed in sperm cells suspended in NCM (99.72 ± 0.09) or CM (98.95 ± 0.12) (Figure 8B). In contrast, NH₄Cl and procaine induced a rapid, concentration-dependent decrease in Lysosensor Green DND-189 signal intensity during the first 10 min of incubation (25 mM NH₄Cl: 79.55 ± 0.27; 2.5 mM procaine: 72.55 ± 0.27; and 10 mM procaine: 36.65 ± 0.44) that was sustained for the entire 60 min incubation (Figure 8B). A slight but significant decrease in Lysosensor Green DND-189 signal during the first 10 min was also observed for sperm cells suspended in CM at pH 7.9 (89.12 ± 0.32); this was also maintained for the rest of the incubation (Figure 8B). Overall, weak bases such as procaine and NH₄Cl increase the pH of acidic cell organelles in stallion sperm in a concentration-dependent fashion whereas high medium pH is much less potent in raising pH of this compartment.

Hyperactivated motility can be reliably induced by 2.5–10 mM procaine

High total motility percentages were observed for sperm cells incubated in 0, 2.5, 5, or 10 mM procaine-containing CM (0 mM: 85 ± 6%; 2.5 mM: 81 ± 6%; 5 mM: 77 ± 4%; 10 mM: 80 ± 2%). Higher procaine concentrations reduced percentages of (total) sperm motility (25: 50 ± 5%; 50 mM: 15 ± 5%) suggesting a procaine concentration-dependent toxic effect on stallion spermatozoa (Figure 9A).

Stallion sperm incubated in CM without procaine showed high percentages of progressive motility (68 ± 7%). Supplementation with procaine markedly reduced percentage of progressively motile sperm at all concentrations tested (2.5 mM: 16 ± 2%; 5 mM: 13 ± 1%; 10 mM: 14 ± 1%; 25 mM: 7 ± 2%; 50 mM: 3 ± 1%) (Figure 9B).

Figure 6.

Combined lightmicroscopic and DNA-fluorescence confocal micrographs of embryo developmental stages (DNA configuration in red) (A and D: 2–4 cell; B and E: 8-cell; C and F: 16-cell) after oocytes were incubated for 18 h in CM + 2.5 mM procaine and CM + 25 mM NH₄Cl, and subsequently cultured in a DMEM/F12 plus 10% FBS based medium. Oocytes that cleaved in conditions containing 2.5 mM procaine and 25 mM NH₄Cl never developed further than the 8–16 cell stage. (original magnification, 400×: Bar = 20 µm).

Maximum ALH and VCL values, indicating hyperactivated motility, were measured when sperm were incubated in 2.5, 5, and 10 mM procaine-supplemented CM (2.5 mM: ALH = 7.3 ± 0.3 µm and VCL = 171 ± 4 µm/s; 5 mM: ALH = 7.2 ± 0.3 µm and VCL = 169 ± 4 µm/s; 10 mM: ALH = 7.1 ± 0.2 µm and VCL = 169 ± 1 µm/s). Much lower ALH and VCL values were observed for sperm cells incubated in CM without procaine (0 mM: ALH = 4.0 ± 0.2 µm and VCL = 102 ± 8 µm/s) or with higher procaine concentrations (25 mM: ALH = 3.2 ± 0.3 µm and VCL = 120 ± 10 µm/s; 50 mM: ALH = 1.0 ± 0.3 µm and VCL = 31 ± 9 µm/s) (Figure 9C and D).

Overall, hyperactivated motility was reliably induced by procaine within the 2.5–10 mM concentration range, whereas higher procaine concentrations were toxic.

Procaine raises cytoplasmic cAMP levels in a concentration-dependent fashion

At 15 min incubation, no significant differences in cAMP levels were measured between sperm suspensions incubated in NCM (0.14 ± 0.09 pM), CM (0.18 ± 0.04 pM), CM without NaCl (0.11 ± 0.03 pM), CM without Na⁺ (0.13 ± 0.10 pM), CM with 25 mM NH₄Cl (0.31 ± 0.03 pM), CM at pH 7.9 (0.27 ± 0.05 pM), and CM with either 2.5, 5, 25, or 50 mM procaine (2.5 mM: 0.14 ± 0.03 pM; 5 mM: 0.41 ± 0.05 pM; 25 mM: 0.50 ± 0.11 pM; 50 mM: 0.52 ± 0.20 pM) (Figure 10B). Significantly higher cAMP concentrations were observed in 10 mM procaine-containing CM (0.77 ± 0.14 pM), while maximum cAMP concentrations were measured for control sperm suspensions incubated in CM plus 1 mM caffeine (1.82 ± 0.22 pM) (Figure 10B). A trend was apparent for a procaine concentration-dependent effect on cAMP concentrations in stallion spermatozoa.

Interestingly, at 15 min incubation, boar sperm demonstrated significantly higher cAMP levels in CM (1.58 ± 0.37 pM) and CM plus 1 mM caffeine (3.95 ± 0.67 pM) than the respective stallion sperm suspensions (CM: 0.18 ± 0.04 pM; CM plus 1 mM caffeine: 1.82 ± 0.22 pM), whereas no significant difference was observed in cAMP levels between boar (0.40 ± 0.19 pM) and stallion (0.14 ± 0.09 pM) sperm suspensions in NCM. Interestingly, cAMP concentrations in boar spermatozoa incubated in CM (1.82 ± 0.22 pM) were similar to those in stallion spermatozoa incubated in CM plus 1 mM caffeine (1.58 ± 0.37 pM) (Figure 10C). For all medium conditions, no significant difference in cAMP concentrations was observed between sperm suspensions incubated for 15 min and 1 h (Figure 10B and 10C). These data indicate that cytoplasmic cAMP in boar sperm incubated in CM is upregulated to a much higher degree than in stallion sperm.

Procaine induces sperm tail-associated protein tyrosine phosphorylation in a concentration-dependent mode

Minimal tail-associated protein tyrosine phosphorylation was observed in NCM (4 ± 2%), CM without NaCl (6 ± 2%), CM without Na⁺ (5 ± 1%), NH₄Cl-supplemented CM (5 ± 2%), and CM with 25 or 50 mM procaine (25 mM: 6 ± 1%; 50 mM: 4 ± 1%). In agreement with other reports [9, 23], maximal percentages of spermatozoa with tail-associated protein tyrosine phosphorylation were observed at pH 7.9 (71 ± 3%). Slightly lower but not different percentages of sperm with protein tyrosine phosphorylation were observed in CM containing 10 mM procaine (53 ± 8%) or caffeine (51 ± 10%). Intermediate tail-associated protein tyrosine phosphorylation incidences were noted after incubation of stallion spermatozoa in both CM (18 ± 4%) and CM plus 2.5 mM procaine (28 ± 4%) (Figure 11). In this experiment, a clear effect of increased medium pH and caffeine on tail-associated protein tyrosine phosphorylation was confirmed, whereas inhibiting Na⁺ influx did not promote this capacitation hall mark. In addition, a procaine concentration-dependent effect on tail-associated protein tyrosine phosphorylation was observed at up to 10 mM procaine.

Figure 7.

Percentages of PD that showed (1) degeneration, (2) meiosis I stage (MI) or meiosis II stage (MII), (3) 1 PN or 2PN, and (4) 1 DNA fragment (1F) or 2 DNA fragments (2F) after 18 h incubation in CM, CM + 2.5 mM procaine, CM—NaCl, CM—Na⁺, CM + 25 mM NH₄Cl, and CM pH 7.9. In general, oocytes exposed to procaine or NH₄Cl rarely formed pronuclei, but instead exhibited condensed DNA fragments. Moreover, high medium pH exerted a degenerative effect on equine oocytes. Data represent mean (±SD) percentages of oocytes after incubation in CM, CM + 2.5 mM procaine, CM lacking NaCl, CM lacking Na⁺, CM + 25 mM NH₄Cl, and CM at pH 7.9; n = 10 oocytes in each group, three replicates. Values that differ significantly (P < 0.05) within degenerated oocytes are indicated by small letters. Values that differ significantly between meiosis I and II oocytes are indicated by capital letters (P < 0.05). Values that differ significantly between oocytes containing DNA fragments are indicated by Greek letters (P < 0.05).

Stallion sperm preincubated in 10 mM procaine capacitating medium do not fertilize equine oocytes in vitro

Oocytes co-incubated in CM with stallion spermatozoa washed after preincubation for 30 min and 4 h in CM, CM + 2.5 mM procaine, and CM + 10 mM procaine did not show oocyte cleavage or cytokinesis at any time point. After sperm had been preincubated for 30 min in CM: 23 ± 6% PD degenerated and 77 ± 5% of parthenogenic division (PD) showed a metaphase plates of meiotic division 1I or II (Mi/MII), in CM + 2.5 mM procaine: 19 ± 8% PD degenerated and 81 ± 5% PD were in MI/MII; and in CM + 10 mM procaine: 27 ± 9% PD degenerated and 73 ± 4% PD were in MI/MII. After 24 h gamete co-incubation, none of the oocytes in any of the groups demonstrated the formation of 2 PN (0 ± 0%) or the presence of a second polar body (0 ± 0%) as evidence of completion of the second meiotic division. Moreover, no differences were observed in any tested group between 0.5 and 4 h sperm preincubation with procaine. These data show that the capacitation-related sperm characteristics induced by 10 mM procaine are not sufficient to support IVF in the horse.