Effects of pH manipulation, CatSper stimulation and Ca²⁺-store mobilization on [Ca²⁺]_i and behaviour of human sperm

Abstract

STUDY QUESTION

How do the alkaline pH, progesterone and 4-aminopyridine interact in their effects on human sperm?

SUMMARY ANSWER

Behaviour of human sperm (proportion of hyperactivated cells and motility kinematics) were related directly to [Ca²⁺]_i irrespective of pH or the agonist applied.

WHAT IS KNOWN ALREADY?

CatSper channels of human sperm, which are central to generation of sperm [Ca²⁺]_i signals and induction of hyperactivated motility, are activated by intracellular alkalinization and progesterone. Progesterone (P4) is much less effective than 4-aminopyridine (4-AP) (which mobilizes stored Ca²⁺ but also raises pH_i) as an inducer of hyperactivation.

STUDY DESIGN, SIZE, DURATION

This was a laboratory study, spanning ~18 months that used 15 sperm donors and involved more than 100 separate experiments.

PARTICIPANTS/MATERIALS, SETTING, METHODS

Semen donors and patients were recruited in accordance with local ethics approval (ERN_12-0570R). [Ca²⁺]_i responses of suspended cell populations were examined by fluorimetric recording and motility parameters assessed by computer-assisted sperm analysis.

MAIN RESULTS AND THE ROLE OF CHANCE

Increasing pH_o from 7.4 to 8.5 raised pH_i (from 6.9 to 7.2) and significantly increased both [Ca²⁺]_i and the proportion of hyperactivated cells. Stimulation of cells with P4 (1 nM–20 μM) induced a biphasic (transient and plateau) increase in [Ca²⁺]_i. The [Ca²⁺]_i increase was of similar amplitude and dose-dependency at pH_o = 7.4 and pH_o = 8.5. 4-aminopyridine (0.2–5 mM) induced a biphasic [Ca²⁺]_i increase that was dose-dependent across the entire range tested and was strongly enhanced at pH 8.5. Motility was assessed 300 s post-stimulation, during the plateau phase of the progesterone and 4-AP-induced [Ca²⁺]_i responses. Progesterone had only a small effect on hyperactivated motility even at the highest dose used (20 μM; < 5% increase in the proportion of cells classified as hyperactivated) which was insensitive to pH_o. 4-Aminopyridine potently stimulated hyperactivated motility, this effect being dose-dependent and greatly enhanced at pH_o = 8.5. The relationship between [Ca²⁺]_i (fluorescence of fluo4) and proportion of hyperactivated cells, irrespective of pH_o, agonist or dose, was fitted by a single curve (second order polynomial; R² = 0.96). Similar analysis of curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) showed a linear relationship to [Ca²⁺]_i (R² > 0.9).

LIMITATIONS, REASONS FOR CAUTION

This was an in-vitro study and caution must be taken when extrapolating these results to in vivo regulation of sperm. Though controls indicate that saturation of fluo4 did not affect the findings, at the highest doses of progesterone the true amplitude of the [Ca²⁺]_i transient may not have been reported by the dye.

WIDER IMPLICATIONS OF THE FINDINGS

These findings indicate that (i) activation of human sperm CatSper by progesterone (and presumably other ligands that act similarly) and consequent acquisition of hyperactivated motility is not significantly enhanced by intracellular alkalinization; (ii) VCL, ALH and hyperactivation are directly related to [Ca²⁺]_i, irrespective of the mechanism by which Ca²⁺ is mobilized, and the ability of stimuli to induce prolonged [Ca²⁺]_i elevation (as occurs upon mobilization of stored Ca²⁺) determines the observed effect on cell behaviour.

STUDY FUNDING/COMPETING INTEREST(S)

CA was supported by the Nigerian government (Tertiary Education Trust (TET) Fund). The authors have no conflicts of interest.

Introduction

Freshly ejaculated mammalian sperm swim with a low-amplitude, symmetrical flagellar beat that generates progressive movement, termed activated motility. Within the female tract (or when incubated under appropriate conditions in vitro) some cells become hyperactivated, a behaviour characterized by exaggerated (often asymmetric) bending of the flagellum which causes greatly increased lateral excursion of the sperm head and may result in continuous turning such that the cell fails to progress (Kay and Robertson, 1988; Suarez, 2008). Hyperactivation is essential for mammalian fertilization. Cells that fail to hyperactivate cannot successfully ascend the female tract or penetrate the zona pellucida (Carlson, et al., 2003; Ho, et al., 2009).

The primary regulatory signal for the transition from activated to hyperactivated motility in sperm of humans (and most mammals where this has been investigated) is an increase in [Ca²⁺]_i (Ho, et al., 2002; Bedu-Addo, et al., 2008; Suarez, 2008). The sperm-specific, Ca²⁺-permeable channel CatSper plays a central role in this process. Sperm of CatSper-null mice fail to hyperactivate and are consequently infertile, and loss of CatSper function in human sperm appears to have similar effects (Carlson, et al., 2003; Smith, et al., 2013; Williams, et al., 2015). CatSper channels are weakly voltage sensitive and are also activated by intracellular alkalinization, which in mouse and bovine sperm may be the key regulator of the channel (Lishko, et al., 2012). Within the female tract both the alkaline environment (which varies both spatially and temporally) and sperm capacitation will increase pHi, regulating the sperm’s behaviour through control of CatSper activity (Cross and Razy-Faulkner, 1997; Fraire-Zamora and Gonzalez-Martinez, 2004; Lishko et al., 2012; Nishigaki, et al., 2014; Ng, et al., 2018). In human (and other primate) sperm the regulation of CatSper appears to be more complex. Though human CatSper is pH sensitive, it is also activated by a wide range of agonists (Lishko, et al., 2011; Strunker, et al., 2011; Brenker, et al., 2012). The best characterized of these, progesterone (P4), occurs at high (micromolar) concentrations in follicular fluid and cumulus but may also be present throughout the tract at concentrations sufficient to regulate activity of the channel (Correia, et al., 2007). In electrophysiological studies stimulation by 500 nM P4 and alkaline pH interacted synergistically (Lishko et al., 2011), but the effects of such interaction on [Ca²⁺]_i and hyperactivation have not been described.

In addition to CatSper-mediated Ca²⁺-influx, regulation of sperm motility by [Ca²⁺]_i can occur through mobilization of stored Ca²⁺, probably from organelle(s) at the sperm neck/midpiece (Ho and Suarez, 2001; Bedu-Addo, et al., 2008). 4-aminopyridine (4-AP), a particularly potent inducer of hyperactivation (Gu, et al., 2004), is a weak base and will stimulate CatSper by raising pH_i, but it has also been shown to mobilize stored Ca²⁺ in a number of cell types including human sperm (Gobet, et al., 1995; Grimaldi, et al., 2001; Bhaskar, et al., 2008; Alasmari, et al., 2013b; Kasatkina, 2016). 4-AP induced hyperactivation in human sperm that were functionally CatSper null (Williams, et al., 2015). When we compared the effects of P4 and 4-AP on motility we found that, even at saturating concentrations, P4 was less effective than 4-AP as an inducer of hyperactivated motility, though it induced a significantly larger [Ca²⁺]_i response (Alasmari, et al., 2013b).

Though it is established that diverse stimuli induce hyperactivation of human sperm via elevation of [Ca²⁺]_i, we know little of how such stimuli interact and combine in their effects on motility. Are the actions of these stimuli simply integrated by [Ca²⁺]_i irrespective of their origin, or does the nature of the original stimulus and/or the source of Ca²⁺ affect the strength of the response? What is the relationship between [Ca²⁺]_i and hyperactivation and do components of hyperactivated motility (increased flagellar excursion causing enhanced lateral head movement, asymmetric beating causing turning/path curvature) show similar [Ca²⁺]_i sensitivity? We have used fluorimetric assay of [Ca²⁺]_i and computer-assisted sperm analysis (CASA) to investigate the interacting effects of P4, 4-AP and elevated pH_i, on [Ca²⁺]_i and hyperactivation in human sperm.

Materials and Methods

Materials, salines

Details of materials and salines (supplemented Earle’s balanced salt solution; sEBSS) are provided in Supplementary Information Materials and Methods.

Ethical approval

Written consent was obtained from donors in accordance with the Human Fertilization and Embryology Authority (HFEA) Code of Practice (version 8) under local ethical approval (University of Birmingham ERC 07-009 and ERN-12-0570).

Selection and preparation of spermatozoa

Semen samples were from donors with normal sperm concentration and motility (WHO 2010). Samples were obtained by masturbation after 2–3 days sexual abstinence. After liquefaction (30 min), sperm were swum up into sEBSS (60 min), adjusted to ≈6 million/ml and left to capacitate (37°C, 6% CO₂) for 5 h (Alasmari et al., 2013b).

Assessment of [Ca²⁺]_i

[Ca²⁺]_i was assessed in fluo4-loaded cells using a FLUOstar microplate reader (BMG Labtech Offenburg, Germany). Details of methodology are provided in Supplementary Information Materials and Methods. Parallel controls with dimethyl sulfoxide (DMSO (vehicle)) at a concentration equivalent to that present in the highest dose used (1% for 4-AP; 0.2% for P4) showed small, inconsistent effects on fluorescence (Supplementary Information Fig. S1a, b). At lower doses (0.00001–0.1%) no effects were detected.

Assessment of pH_i

pH_i was assessed in 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF, AM)-loaded cells using a FLUOstar microplate reader (BMG Labtech Offenburg, Germany). Details of methodology, measurement and calibration are provided in Supplementary Information Materials and Methods.

Assessment of motility

Motility of sperm samples was assessed at 35°C using a Hamilton Thorn CEROS CASA system. Details of methodology are provided in Supplementary Information Materials and Methods.

Statistical analysis

Statistical significance was determined using Student’s paired/unpaired t-test or ANOVA and adjusted using the Holm–Bonferroni correction for multiple comparisons (Gaetano, 2013) as appropriate. Percentage data were transformed using the arcsine square root conversion (Sokal and Rohlf, 1981) before statistical analysis to allow application of parametric tests. Data are presented as mean ± SEM. Unless stated otherwise, the values of ‘n’ for [Ca²⁺]_i and motility assessments provided in text and figure legends show the number of experiments used for statistical analysis.

Results

pH_o and pH_i

To assess the effect of manipulating extracellular pH on pHi, BCECF-loaded sperm were suspended in media buffered to a range of pH values (6.0–9.0) then exposed to 0.12% Triton X-100 (Fraire-Zamora and Gonzalez-Martinez, 2004), allowing BCECF ratios to be recorded both from intact cells and after permeabilsation to equilibrate pHi with pH_o (Supplementary Information Fig. S2a). Using a calibration curve for BCECF fluorescence obtained from permeabilised cells (Supplementary Information Fig. S2b) we estimated pH_i for intact cells. As described previously (Hamamah et al., 1996), pH_i was strongly correlated with pH_o (Supplementary Information Fig. S2c). To investigate the effects of pH on [Ca²⁺]_i signalling and motility in human sperm we selected values for pH_o of 7.4 and 8.5 (pH_i = 6.85 ± 0.06 and 7.19 ± 0.14, respectively; n = 5 experiments; P = 0.02; Fig. 1a). The proportion of motile cells was slightly greater at pH_o = 8.5 (Fig. 1b; P < 0.05) and the proportion of progressively motile was not affected (Fig. 1c). In contrast, both resting [Ca²⁺]_i and hyperactivated motility (% hyperactivated cells) were markedly increased at pH_o = 8.5 compared to pH_o = 7.4 (Fig. 1d and e; P < 10⁻⁵ and P < 10⁻⁶, respectively).

Interaction of progesterone and pH

When P4 was applied to cells suspended in standard sEBSS (pH_o = 7.4) we observed an increase in [Ca²⁺]_i that peaked within 20 s, decayed over the following 60–90 s and was followed by a plateau phase which was maintained for the duration of recording (300 s; Fig. 2a, left panel). Both transient and plateau saturated at 0.1–1 μM P4 and were clearly detectable (and statistically significant compared to the preceding control period) when the cells were stimulated with doses as low as 1 nM (Fig. 2b and c; grey bars). In parallel experiments carried out with cells that had been suspended in saline buffered to pH 8.5, P4 induced a similar biphasic [Ca²⁺]_i elevation though the decay of the transient was clearly slower under these conditions (Fig. 2a, right panel). P4-induced transient and plateau responses were both dose dependent (P = 1.3 × 10⁻⁹ and 2.7 × 10⁻⁶ for transient and plateau, respectively) but there was no significant effect of pH_o (P = 0.12 and 0.83 for transient and plateau, respectively, 2-way ANOVA; Fig. 2b and c). Under both conditions the transient and plateau responses to P4 (20 μM) significantly exceeded those seen in parallel controls where vehicle (DMSO) was applied at equivalent concentration (Fig. 2b and c)

The failure of elevated pH to enhance the increment in fluorescence induced by P4 concentrations ≥0.1 μM might reflect limitations of the assay. If saturation of fluo4 occurs during the [Ca²⁺]_i transient the dye would fail to report any enhancement of the [Ca²⁺]_i signal at pH_o = 8.5. To investigate this we carried out further experiments in which cells, suspended at pH_o = 8.5, were first stimulated with P4 (0.001–20 μM) then 10 μM ionomycin was added 30–40 s after the peak of the P4-induced [Ca²⁺]_i transient. In six experiments ionomycin consistently increased fluorescence above the levels induced by P4, the size of the maximum fluorescence increase being 40–50% greater than that reached during the [Ca²⁺]_i transient induced by saturating concentrations of P4 (P < 0.01; Supplementary Information Fig. S3a, b). Though this suggests that our failure to observe an enhanced [Ca²⁺]_i response to P4 at pH_o = 8.5 was not due to dye saturation, we further investigated this by assessing responses to 0.1 μM and 1 μM P4 using a lower affinity dye (Fluo5F), which has a reported in vitro K_d of 2.35 μM compared to 345 nM for fluo4 (6.8-fold difference; Gee et al., 2000). There was now a clear difference in the amplitude of the P4-induced transient induced by these two doses of P4 (P < 0.005; Supplementary Information Fig. S3d), which was not apparent when using fluo4. However, we still observed no significant enhancement of the P4-induced [Ca²⁺]_i signal at pH_o = 8.5 (P > 0.5; Supplementary Information Fig. S3c, d).

We next examined the effect of pH_o on the ability of P4 to induce hyperactivated motility. Stimulation of sperm suspended in standard sEBSS with P4 (0.1–20 μM) induced a small (<5%) but significant increase in the proportion of hyperactivated cells that was dose-independent over the range used (Fig. 2d; grey bars). In parallel experiments with cells suspended at pH_o = 8.5 the effect was of similar amplitude (Fig. 2d; black bars). Analysis of the data by 2-way ANOVA confirmed that neither the effect of P4 dose nor the effect of pH_o was significant (P = 0.72 and 0.89, respectively).

Interaction of 4-AP and pH

Similarly to the effect of P4, application of 4-AP to cells suspended in standard sEBSS (pH_o = 7.4) induced a biphasic increase in [Ca²⁺]_i (Alasmari et al., 2013a). The initial transient reached a maximum after 20–30 s, decayed over the following 50–60 s and was then followed by a plateau which was maintained for the duration of recording (300 s; Fig. 3a, left panel). The amplitudes of both transient and plateau (300 s post-stimulation) were dose-dependent (P = 6 × 10⁻⁶ and P = 2.0 × 10⁻⁵, respectively; 1-way ANOVA). [Ca²⁺]_i elevation was clearly detectable at the lowest concentration of 4-AP tested (0.2 mM; P < 0.01 compared to preceding control period), but this effect apparently saturated at 0.4–0.6 mM, a further enhancement occurring at concentrations >1 mM (Fig. 3b and c, grey bars). Both transient and plateau responses to 4-AP (5 mM) significantly exceeded those seen in parallel controls where vehicle (DMSO) was applied at equivalent concentration (Fig. 3b and c; grey bars).

Application of 4-AP to cells suspended at pH_o = 8.5 induced dose-dependent [Ca²⁺]_i responses similar to those seen at pH 7.4 though the amplitudes of both transient and sustained responses were significantly greater (P = 6.9 × 10⁻⁴ and P = 1.9 × 10⁻⁶, respectively; 2-way ANOVA; Fig. 3). As with responses to P4, decay of the [Ca²⁺]_i transient was slower at the higher pH_o (Fig. 3a). Whereas the sustained response induced by P4 was relatively small and of consistent amplitude relative to the preceding transient (25–30% at 300 s post-stimulation; Fig. 3e; yellow symbols), in 4-AP-stimulated cells the sustained response became larger (relative to the preceding transient) as the dose was increased (Fig. 3b and c; Fig. 3e; black symbols). When cells suspended at pH 8.5 were stimulated with the highest dose of 4-AP (5 mM) we observed no decay of the [Ca²⁺]_i signal after the initial peak (Fig. 3a, right panel).

Stimulation with 4-AP of cells suspended in standard sEBSS (pH 7.4) had little effect on motility at doses of 0.2–0.6 mM but with concentrations of 4-AP ≥0.8 mM we observed a significant and dose-dependent increase in the proportion of hyperactivated cells (Fig. 3d; P < 0.05). When cells were suspended at pH_o = 8.5 significant stimulation of hyperactivated motility was seen at all doses tested (0.2–5 mM) and the effect was strongly dose dependent (Fig. 3d). Analysis of the data by 2-way ANOVA confirmed that both dose-dependence and pH_o sensitivity were highly significant (P = 1.7 × 10⁻¹² and 1.5 × 10⁻²¹, respectively). Motility (% cells) was not affected by exposure to 5 mM 4-AP for 300 s, either at pH_o = 7.4 or pH_o = 8.5 (P > 0.3).

Is the effect of 4-AP due to cytoplasmic alkalinization?

4-AP is much more potent than P4 in stimulating hyperactivation of human sperm (demonstrated in this study, see Fig. 2d and 3d and our previous studies; Alasmari et al., 2013a,b). Previously we established that, in cells suspended in standard sEBSS (pH 7.4) this effect was not due merely to the ability of 4-AP (a weak base) to raise pH_i, since 25 mM NH₄Cl, which had a similar effect on cytoplasmic pH_i, had only modest effects on motility (Alasmari et al., 2013b). During this study we carried out similar experiments on cells suspended in saline buffered to pH 8.5. Exposure of cells to 2 mM 4-AP for 300 s increased pHi by ≈0.5 units and in parallel experiments the effect of 25 mM NH₄Cl on pH_i was greater (this difference was NS; Supplementary Information Fig. S4a). In contrast, the effect of 2 mM 4-AP on the proportion of hyperactivated cells was significantly greater than that of NH₄Cl, which had negligible effects on motility (Supplementary Information Fig. S4b).

Relationship between [Ca²⁺]_i and motility in sperm stimulated with P4, 4-AP and high pHo

Stimulation of human sperm with P4 or by cytoplasmic alkalinization elevates [Ca²⁺]_i and modifies motility primarily by activation of CatSper (Lishko et al., 2012) whereas the more potent effects of 4-AP involve mobilization of stored Ca²⁺ in addition to alkalinization (see Introduction). To compare the relative efficacies (regulation of sperm behaviour) of the [Ca²⁺] signals induced by P4 and 4-AP (and the effects on these of pH) we plotted, for each stimulus protocol (agonist concentration and pH), the mean fluo4 fluorescence intensity at 300 s post-stimulation (time of CASA data collection) and mean values for CASA motility parameters (hyperactivation, VCL, ALH and LIN). Figure 4a shows the relationship between fluorescence intensity and % hyperactivated cells for four concentrations of P4 (0.1–20 μM; yellow symbols; controls shown green) and seven concentrations of 4-AP (0.2–5 mM; grey symbols; controls shown black). Experiments at pH_o = 7.4 and 8.5 are plotted as circles and triangles, respectively. Though the cells used for the P4 experiments showed slightly higher levels of ‘spontaneous’ hyperactivation than those used during 4-AP experiments (compare green and black control symbols), the points describe a single curve best fitted by a second order polynomial (y = 0.009ײ − 0.57× + 8.6; R² = 0.96; Fig. 4a). Figure 4b–d shows equivalent plots for VCL, ALH and LIN, the three kinematic parameters used to define hyperactivation in CASA analysis. Both VCL (Fig. 4b) and ALH (Fig. 4c) were linearly related to fluo4 fluorescence (R² = 0.93 and 0.91, respectively). The relationship between fluo4 fluorescence and LIN was more complex. The data from 4-AP experiments fell on a single curve (second order polynomial; R² = 0.97) but, in cells stimulated with P4, values for LIN were markedly lower (Fig. 4d, yellow symbols). LIN is calculated from the ratio of VSL:VCL (blue:black lines in Fig. 5b) so this effect of P4 could reflect increased lateral deviation of the sperm head (ALH) and/or greater curvature of the average path (red line in Fig. 5b). Since ALH values from the P4 and 4-AP experiments clearly lie on the same line (Fig. 4c), this suggests that P4 particularly increases path curvature. We therefore assessed straightness (STR; the ratio VSL:VAP; blue:red lines in Fig. 5b), which is determined primarily by path curvature. Low doses of P4 (0.1–1 μM) significantly reduce STR, similarly to their effect on LIN (compare Fig. 4d and 5a). This effect was particularly marked at pH_o = 8.5 (Fig. 5c). 4-AP had an equivalent effect on STR only at the highest doses (1–5 mM; Fig. 5a and d), when [Ca²⁺]_i reached levels exceeding those seen with P4 stimulation. Thus, at equivalent [Ca²⁺]_i, P4 increased path curvature more than 4-AP.

Discussion

Mammalian sperm ascending the female tract experience an increase in pH from ≈4 in the vagina to ≈8.0 in the oviduct (Ng, et al., 2018). As reported previously (Hamamah et al., 1996), pH_i of human sperm was sensitive to pH_o. Surprisingly, though both [Ca²⁺]_i and hyperactivated motility were significantly enhanced by increased pH_o, consistent with activation of CatSper by alkalinization, the effects of P4 (1 nM–20 μM) on [Ca²⁺]_i and motility showed negligible pH-sensitivity. Fraire-Zamora and Gonzalez-Martinez (2004) reported a similar lack of effect when using NH₄Cl to alkalinize the cytoplasm. Control experiments indicated that saturation of fluo4 did not ‘hide’ enhanced [Ca²⁺]_i transients and, more importantly, during the P4-induced [Ca²⁺]_i plateau recorded at pH_o = 8.5 fluo4 was no more than 65% saturated, yet the P4-induced [Ca²⁺]_i-increment was similar at pH_o = 7.4 and 8.5. We conclude that increased pH_o and consequent cytoplasmic alkalinization does not significantly enhance Ca²⁺-mediated modulation of motility by P4. Thus, though the more alkaline pH of the upper female tract will modulate CatSper, increasing [Ca²⁺]_i and changing the behaviour of the sperm, sensitivity to the high concentrations of P4 encountered in the vicinity of the cumulus–oocyte complex may be unchanged.

Similar experiments with 4-AP showed significant dose- and pH-dependent effects on both [Ca²⁺]_i and motility. At pH_o = 7.4 we observed a small though significant effect on [Ca²⁺]_i which saturated at 0.2–0.6 mM and at higher doses there was a second and much greater effect on both [Ca²⁺]_i and hyperactivation. This complex effect is not surprising since 4-AP may both activate CatSper by raising pH_i and mobilize stored Ca²⁺ at doses as low as 0.25 mM (Alasmari et al., 2013b; Kasatkina; 2016). Chavez, et al. (2018) have recently shown that alkalinization of the acrosome by weak bases (though 4-AP was not tested) induces release of stored Ca²⁺. At pH_o = 8.5 4-AP was effective even at low doses, probably because alkaline pH favours the non-ionized (cell-permeant) form of 4-AP, facilitating intracellular accumulation of the drug (Howe and Ritchie, 1991).

P4-induced hyperactivation of human sperm in vitro has been reported on a number of occasions and is clearly illustrated in the videos of Smith, et al. (2013) and Schiffer, et al. (2014), yet in this study (and others; Alasmari et al., 2013a,b) the potency of P4 compared to 4-AP was negligible. This inconsistency may reflect the kinetics of the [Ca²⁺]_i responses in these in vitro experiments, where agonists are applied as a bolus. P4-induced [Ca²⁺]_i transients are large but decay rapidly such that when motility is assessed 300 s post-stimulus the effect of P4 is negligible. In contrast, the 4-AP-induced signal decays little, particularly at high doses, so that persistent and potent stimulation of hyperactivation is observed. We hypothesize that this difference reflects 4-AP-induced Ca²⁺ store mobilization (see above) and consequent activation of store-operated channels (Lefievre, et al., 2012). In a minority of P4-stimulated cells CatSper-mediated Ca²⁺-influx induces [Ca²⁺]_i oscillations superimposed on the P4-induced [Ca²⁺]_i-plateau, an effect which apparently reflects secondary release of stored Ca²⁺ (Harper et al., 2004). Such [Ca²⁺]_i-oscillations may be required for prolonged motility regulation by P4 (Bedu-Addo et al., 2008) and may underlie the repeated switching between activated and hyperactivated behaviours seen in human sperm (Publicover, 2017).

To investigate the relationship between pH, agonist type/concentration, [Ca²⁺]_i, and sperm behaviour we calculated mean [Ca²⁺]_i (absolute fluorescence intensity) and CASA kinematics for each condition for which equivalent fluorimetric and CASA data had been collected (22 in total). Plotting of the relationship between [Ca²⁺]_i and hyperactivation produced a curve that was best fitted by a second order polynomial (R² = 0.96). Data fell on this curve irrespective of pH_o or agonist type. Similar analysis of VCL and ALH (two of the kinematics used to define hyperactivation) generated linear plots (R² > 0.91). These data clearly suggest that both curvilinear velocity and lateral movement of the sperm head are determined primarily by [Ca²⁺]_i, irrespective of pH or the nature of the agonist. The values of VCL and ALH used to define hyperactivation (150 μm s⁻¹ and 7 μM, respectively; Mortimer, 2000) both occurred at a fluorescence intensity of ≈90 000 (Fig. 4; red dashed lines). Data for LIN from 4-AP experiments (but not P4, see below) were fitted by a second order polynomial, the hyperactivation threshold value of 50% again occurring at ≈90 000. To obtain a rough estimate of [Ca²⁺]_i we used the data from Harper et al. (2003), who calibrated their ratiometric fura2 recordings from human P4-stimulated sperm populations prepared and treated (apart from recording at 37°C) exactly as described here. Using the P4-induced [Ca²⁺]_i transient as a bioassay (Supplementary Information figure S5), we estimate that [Ca²⁺]_i corresponding to a fluorescence of ≈90 000 (hyperactivation ‘threshold’ values for VCL/ALH/LIN) is 600–700 nM and that 50% hyperactivation (50% of cells satisfy all three kinematic criteria) occurs at ≈800 nM. In re-activated, ‘skinned’ bovine sperm a [Ca²⁺] of ≈200 nM induced 50% hyperactivation and the effect saturated at 400 nM (Ho et al., 2002).

Plotting of values for LIN and STR (straightness, which assesses the curvature of the sperm’s average path) against [Ca²⁺]_i showed striking differences between cells stimulated with P4 and with 4-AP. At equivalent levels of fluo4 fluorescence, whereas LIN and STR were markedly reduced in P4-stimulated cells, 4-AP had no effect (in fact a non-significant increase in STR was recorded). Control values of LIN and STR in P4 experiments were lower than those in experiments with 4-AP (particularly at pH_o = 7.4) and this observation should therefore be interpreted cautiously. However, this striking difference in the effects of P4 and 4-AP is not apparent in the data for VCL or ALH. One possibility is that P4 has ‘extra’, non-[Ca²⁺]_i-dependent effects on curvature of the cell path. For instance, activation of Erk1/2, p90RSK, p38MAPK by P4 (Sagare-Patil, et al., 2012) may affect motility. This unexpected effect of P4 on motility deserves further investigation since induction of turning without other characteristics of hyperactivated motility may play a role in enabling sperm to locate the oocyte.

In summary, our data indicate a clear relationship between [Ca²⁺]_i and hyperactivation that is independent of pH or the mechanism of agonist-induced Ca²⁺-mobilization. 4-AP, which mobilizes stored Ca²⁺, is more effective in promoting persistent elevation of [Ca²⁺]_i and consequent modulation of sperm behaviour and Ca²⁺-store-dependent mechanisms may, therefore, be important in tonic regulation of motility in vivo.

Acknowledgements

None.

Authors’ roles

C.A., V.P. and R.T. carried out the laboratory work. C.A. and S.P. analysed the data and prepared the article.

Funding

C.A. was supported by the Nigerian government (Tertiary Education Trust (TET) Fund).

Conflict of interest

The authors have no conflicts of interest.

References

Author notes