Zn²⁺ induces hyperpolarization by activation of a K⁺ channel and increases intracellular Ca²⁺ and pH in sea urchin spermatozoa

Abstract

Zinc (Zn²⁺) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn²⁺ was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pH_i). In this study we found that submicromolar concentrations of free Zn²⁺ change membrane potential (Em) and increase the concentration of intracellular Ca²⁺ ([Ca²⁺]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn²⁺ response in sperm of this species mainly involves an Em hyperpolarization caused by K⁺ channel activation. The pharmacological profile of the Zn²⁺-induced hyperpolarization indicates that the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn²⁺. Considering that Zn²⁺ also induces [Ca²⁺]i fluctuations, our observations suggest that Zn²⁺ activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn²⁺ in male gamete function.

Introduction

Although Zn²⁺ is a trace element, it is known to be essential for a wide range of biological processes: transcription, cell division, proliferation, immune function, defense against free radicals and fertilization (Pluth et al., 2011). In mammals, it has been known for long time that Zn²⁺ concentration in the seminal fluid is notably high (Hidiroglou and Knipfel, 1984; Mawson and Fischer, 1953). In fact, the highest concentration in the human body is in the seminal plasma (3 mM; (Saaranen et al., 1987)). Zn²⁺ has also been proposed to stabilize chromatin (Bjorndahl and Kvist, 2010) and regulate sperm capacitation (Andrews et al., 1994; Aonuma et al., 1978; Lishko et al., 2010). Furthermore, it was recently reported that Zn²⁺ is also important for male gamete motility regulation in the nematode C. elegans (Liu et al., 2013).

Upon dilution of sea urchin sperm in seawater, metal chelators such as EDTA, EGTA, phenantroline and cysteine, at concentrations that do not alter the Ca²⁺ or Mg²⁺ levels (0.1-1 mM), delay motility initiation and inhibit the AR induced by egg jelly (Clapper et al., 1985; Johnson and Epel, 1983). Among the trace ions in seawater (such as Cu²⁺ and Ni²⁺), only Zn²⁺ can rescue these chelator inhibitory effects, indicating a crucial role of this divalent cation in sea urchin sperm physiology. In addition, Zn²⁺ is able to induce the AR of Lytechinus pictus spermatozoa, although its physiological relevance is unknown. The action of Zn²⁺ in sea urchin spermatozoa has been attributed to pH_i regulation (Clapper et al., 1985).

It is well established that echinoderm spermatozoa respond to sperm-activating peptides (SAPs) derived from the egg jelly of the same species which regulate motility (Suzuki, 1995). Speract is the first structurally identified SAP from the egg jelly of Hemicentrotus pulcherrimus (Suzuki et al., 1981) and Strongylocentrotus purpuratus (Hansbrough and Garbers, 1981), and also stimulates L. pictus spermatozoa (Guerrero et al., 2010; Nishigaki and Darszon, 2000). Speract binding to its receptor in the flagella plasma membrane stimulates the synthesis of cGMP (Garbers, 1989) which directly activates the cGMP-gated K⁺ selective channel (tetraKCNG/CNGK) inducing a K⁺ efflux and a membrane hyperpolarization (Babcock et al., 1992; Bonigk et al., 2009; Galindo et al., 2007). This hyperpolarization leads to increases in pH_i (Lee, 1984), [Ca²⁺]i, and cAMP levels (Beltran et al., 1996). All these changes induced by speract, except for the cGMP increase, are inhibited by elevating the K⁺ concentration in seawater (Harumi et al., 1992).

In this study we show that submicromolar free Zn²⁺ concentrations induce similar sperm responses as speract does including Em changes, cAMP elevation and [Ca²⁺]_i fluctuations except for the cGMP increase in L. pictus spermatozoa. We discuss the molecular mechanisms and physiological significance of Zn²⁺ for sea urchin spermatozoa.

Material and Methods

Gametes and reagents

L. pictus sea urchins were obtained from Marinus (Long Beach, CA, USA). Dry sperm were collected after intracelomic injection of 0.5 M KCl and kept on ice until used. The fluorescent dyes 3,3'-dipropylthiadicarbocyanine iodide (DiSC3(5)), Fluo-4-AM, Quin-2 and 5-(and-6)-Carboxyfluorescein diacetate were obtained from Molecular Probes (Eugene, OR, USA). Anhydrous dimethylsulfoxide (DMSO), tolbutamide, glibenclamide, were from Sigma-Aldrich. ZnSO₄ was from Merck. Charybdotoxin and Iberiotoxin were from Alomone Labs. The Kits to measure cAMP (TRK 432) and cGMP (TRK 500) were from Amersham. Speract was synthesized in Professor Possani's Laboratory (IBT-UNAM) and fucose sulfate polymer (FSP) was prepared according to the previous report (Garbers et al., 1983). The rest of the reagents used were of the highest quality available.

Composition of artificial seawater (ASW)

Normal ASW was prepared with the following composition (in mM): 465 NaCl, 26 MgCl₂, 10 KCl, 30 MgSO₄, 10 CaCl₂, 2.5 NaHCO₃ and 0.1 EDTA (or otherwise indicated) with pH 8.0 by NaOH. Ca²⁺-free pH 8.0 (0CaSW) or 1 mM Ca²⁺ pH 7.0 (1CaSW) ASWs were the same except for the indicated CaCl₂ concentration and pH. High K⁺ ASW (50KSW) contained 50 mM KCl. Cl^--free ASW (0ClSW) was prepared by substituting NaCl and KCl with each metanesulphonate salt and substitution of MgCl₂ and CaCl₂ with each sulfate salt. In all cases the osmolarity was 950-1000 mOsm.

Fluorescence measurements with sperm suspension

Fluorometric measurements were performed in an OLIS-upgraded SLM 8000 Aminco spectrofluorometer with a temperature-controlled cell holder (14 °C) and a magnetic stirrer. Spermatozoa [Ca²⁺]_i and pH_i measurements were done according to Rodríguez and Darszon (Rodriguez and Darszon, 2003). Briefly, diluted (1:5) dry sperm in 1CaSW containing 20 μM of Quin-2-AM or Fluo 4-AM without or with 0.5 % Pluronic F-127 or 10 μM Carboxyfluorescein diacetate were incubated for 3 h at 14 °C in the dark. After loading with these fluorescent dyes, 10 ml of 1CaSW were added to the sperm suspension. Coelomocytes and spines were precipitated by mild centrifugation (121 g for 7 min at 4 °C) and the sperm suspension was applied to further centrifugation (1000 g for 8 min at 4 °C) to eliminate the dyes remaining in the media. The sperm pellet was resuspended in the original volume of 1CaSW and kept on ice in the dark until used. A 10 μl aliquot of the loaded sperm was added to a flat-bottom glass tube containing 800 μl ASW. After 30 seconds, an agonist was added using a Hamilton syringe. Fluorescence intensities (excitation/emission wavelength) for Quin-2 (340/490 nm), Fluo-4 (505/525 nm) and Carboxyfluorescein diacetate (490/535 nm) were recorded every 0.5 s.

For Em experiments, 10 μl of diluted (1:10 in 1CaSW) sperm were added to 800 μl of ASW plus 0.7 mM EDTA and 0.5 μM DisC3(5). The EDTA concentration for Em measurements was increased because DisC3(5) induces the acrosomal reaction in ASW with 0.1 mM EDTA, but not in 0.8 mM EDTA. Fluorescence intensities of DisC3(5) (640/670 nm) were registered every 0.5 s. After 2-3 minutes, a protonophore (CCCP 1 μM) was added to avoid mitochondrial membrane potential interference. Once the fluorescence reached the equilibrium, an agonist was added. DisC3(5) fluorescence was calibrated using 1 μM valinomycin and sequential additions of KCl. Em was calculated according to the Nernst equation assuming that the intracellular K⁺ concentration is 180 mM (Babcock et al., 1992). For fluorometric experiments, stock solutions of Fluo-4-AM (1 mM) and Carboxyfluorescein diacetate (1 mM) and DiSC3(5) (0.8 mM) were made in anhydrous DMSO.

Single cell fluorescence imaging

Fluorescence imaging was carried out as previously reported (Nishigaki et al., 2004). Briefly, Fluo-4-labeled spermatozoa were adhered to glass coverslips coated with 50 Pg/ml poly-L-lysine solution (Sigma) and mounted into a micro incubator, PDMI-2 (Harvard Apparatus, Holliston, MA, USA) maintained at 14 °C. Fluorescence images were acquired using a Nikon DIAPHOT 300 inverted microscope with a Nikon Plan Apo 60X objective lens (1.4 NA) and a custom-built stroboscopic illumination system (Nishigaki et al., 2006) with a Chroma filter set (Ex, HQ470/40x; DC, 505DCXRU; Em, HQ510LP (Chroma Technology)). Images were acquired with a Quantix 57 camera (Photometrics Inc.) under the continuous (stream) acquisition mode.

Determination of cAMP and cGMP levels

Sperm diluted (1:200) in 0CaSW with 1 mM 3-isobutyl-1-methylxanthine (IBMX) were incubated with a ligand (Zn²⁺ or speract) for 1 min at 14 °C. Subsequently, the sperm suspension was transferred to a boiling bath to quench the enzymatic reaction and extract nucleotides. TRK 432 or TRK 500 Kits of Amersham were used to measure cAMP or cGMP concentration, respectively.

Estimation of free Zn²⁺ concentration

The free Zn²⁺ concentration in ASW after various Zn²⁺ additions was estimated using a WinMaxC 2.4 software package (Stanford University, Chris Patton http://www.stanford.edu/~cpatton/downloads.htm). DisC3(5) contains a certain amount of Zn²⁺ as a contaminant and though small amount of DisC3(5) (0.5 PM) should not significantly affect the total Zn²⁺, in most Em experiments ASW containing 0.8 mM EDTA was used. Addition of 10 μM ZnSO₄ to ASW containing 0.8 mM EDTA yields ~ 0.2 nM free Zn²⁺.

Data normalization

Although the absolute changes in the sperm Em obtained within one season are very reproducible, there is variability between experiments with cells from different seasons. For this reason we normalized the data in order to compare them. Furthermore, in experiments where high K⁺ (50KSW) was used to block the speract responses it is difficult to calibrate Em using the Nernst equation. Therefore, Em changes in 50KSW were expressed with F/F₀, where F is fluorescence intensity of DisC3(5) and F₀ is a F value just before addition of Zn²⁺.

Statistics

Student's t test or one-way ANOVA were used for statistical evaluations. Differences among experimental conditions were considered significant when p < 0.05.

Results

Zn²⁺ changes membrane potential (Em) and increases pH_i and [Ca²⁺]_i

It is known in sea urchin sperm that Zn²⁺ increases pH_i (Clapper et al., 1985). Since Zn²⁺ is also known to induce the AR in L. pictus spermatozoa, we determined changes of Em, pH_i, and [Ca²⁺]_i triggered by Zn²⁺ as well as those induced by FSP, the natural AR inducer, and speract. The upper records in Figure 1 show that addition of 10 μM Zn²⁺ (0.2 nM free Zn²⁺), FSP (~100 nmol fucose/ml) and 100 nM speract causes a transient Em hyperpolarization followed by a depolarization. The Zn²⁺-induced Em changes are different from those triggered by FSP (middle upper trace), but rather similar to those induced by speract. The three inducers altered pH_i in a somehow similar fashion (center traces). While the [Ca²⁺]_i increase induced by speract is a transient, those induced by Zn²⁺ and FSP are relatively permanent probably due to the induction of AR (lower records). It is worth noting that the FSP induced [Ca²⁺]_i changes presented in Fig. 1B were performed using Quin-2 (see Methods) and thus appear smaller than those induced by Zn²⁺ and speract where Fluo-4 was used.

Fig. 1.

Zn²⁺ changes Em and increases pH_i and [Ca²⁺]_i as FSP and Speract.

Em (upper row records), pHi (middle row records) and [Ca²⁺]_i (lower row records) of sea urchin sperm were measured in cell populations as described in methods. Dotted lines indicate a point of agonist addition: A, Zn²⁺ (10 M total); B, FSP (~100 nmol fucose/ml); C, 100 nM Speract. The free concentration of Zn²⁺ was estimated to be 0.2 nM in Em measurement (0.8 mM EDTA) and 1.8 nM in pH_i and [Ca²⁺]_i measurements (0.1 mM EDTA). Downward and upward deflections of each parameter indicate hyperpolarization and depolarization of Em and decreases and increases of [Ca²⁺]_i and pH_i, respectively. The scale of each parameter Em (mV), pH_i (FAU, Fluorescence Arbitrary Units) and [Ca²⁺]_i ((F/F0); where F is Fluo-4 (or Quin-2) fluorescence intensity and F0 is the fluorescence before adding speract or Zn²⁺) is indicated with vertical bars. The time scale was indicated by horizontal bars. Records shown in this figure are representative of at least three different animals. Zn²⁺ and FSP induced similar amounts of acrosome reaction that were ≥57%.

Zn²⁺hyperpolarizes L. pictus sperm through K⁺ channel activation

As a next step, we measured Em changes with different concentrations of Zn²⁺. Figure 2A shows representative records of Em changes induced by different concentrations of Zn²⁺ and Figure 2B, the average of Em hyperpolarization. Using the results from Figure 2B, we estimated a DL₅₀ of Zn²⁺ for the L. pictus sea urchin sperm hyperpolarization of 0.38 μM.

Fig. 2.

Zn²⁺ hyperpolarizes the sperm Em in a dose dependent manner.

Representative traces of the sperm Em changes triggered by different Zn²⁺ concentrations (0.05-300 μM) are shown in panel A. Each number indicates the total concentration of Zn²⁺ in PM and the estimated free Zn²⁺ concentration is in parenthesis. To avoid trace confusion, black and gray lines were used alternatively. Panel B shows the best fit curve of the hyperpolarization change as a function of log[Zn²⁺] for three different animals with triplicate measurements. One way ANOVA was used to examine if the differences between different Zn²⁺ concentrations was significant at *p < 0.05.

A simple mechanism for the Em hyperpolarization might be the activation of electrogenic K⁺ efflux or Cl^- influx. Therefore, we explored these two possibilities by altering the ionic compositions of artificial seawater. As shown in Figure 3A, high K⁺ ASW (50KSW) effectively inhibited the Em hyperpolarization induced by Zn²⁺, although the subsequent Em depolarization was not affected significantly. In contrast, Cl^--free ASW (0ClSW) had a small effect on Em hyperpolarization induced by Zn²⁺ (Fig. 3B). These results suggest that one of the principal targets for Zn²⁺ may be a K⁺ channel of sea urchin spermatozoa.

Fig. 3.

High K⁺ ASW (50KSW) inhibits the Zn²⁺-induced hyperpolarization.

Panel A shows representative sperm Em responses to 10 μM Zn²⁺ (0.2 nM free Zn²⁺) in normal ASW (black line) and in 50KSW (gray line). Panel B shows the magnitude of Em hyperpolarization (mean ± SE) induced by 10 μM Zn²⁺ in 50KSW and Cl^- free ASW (0ClSW). The magnitude of hyperpolarization (delta F/F₀) was normalized against control experiments performed in normal ASW. Statistically significant differences are indicated by asterisks (n = 5 for 50KSW and n = 4 for 0ClSW; *p 0.05).

Effects of K⁺ channel blockers on Zn²⁺-induced Em hyperpolarization

In order to determine the type of K⁺ channel involved in the Zn²⁺-induced Em changes, we treated the spermatozoa with several K⁺ channel blockers. As seen in Figure 4A, 20 mM of TEA (tetraethylammonium), which blocks many types of K⁺ channel including one activated by FSP (Guerrero and Darszon, 1989), did not inhibit the Zn²⁺-induced hyperpolarization. It was reported that Zn²⁺ activates K_ATP channels in pancreatic beta cells and brain nerve terminals (Bancila et al., 2005; Bloc et al., 2000; Matias et al., 2010; Prost et al., 2004), but two K_ATP channel blockers, either tolbutamide (Fig. 4B) or glibenclamide (Fig. 4C), did not inhibit the Zn²⁺-induced hyperpolarization. Moreover, neither Charybdotoxin, a blocker of BK/Slo1 (Gimenez-Gallego et al., 1988; Miller et al., 1985) and Kv1 K⁺ channels (Grissmer et al., 1994), nor Iberiotoxin another blocker for BK/Slo1 K⁺ channel (Galvez et al., 1990) inhibited the Zn²⁺-induced hyperpolarization (Fig. 4D). Notably, none of these K⁺ channel inhibitors were able to significantly decrease the magnitude of the Zn²⁺ induced Em hyperpolarization; instead some reagents rather enhanced it (Fig. 4E). These results better correspond to the pharmacological profile of tetraKCNG/CNGK, which is key to the speract signaling cascade, reported before (Bonigk et al., 2009; Galindo et al., 2007).

Fig. 4.

The Zn²⁺-triggered hyperpolarization is not inhibited by several K⁺ channel blockers. Traces A-D indicate representative Em changes induced by 10 μM Zn²⁺ (0.2 nM free Zn²⁺) in ASW containing 20 mM tetraethylammonium (TEA) in panel A, 300 μM Tolbutamide (Tolbu) in panel B, 20 μM Glibenclamide (Glib) in panel C, or 100 nM Charybdotoxin (ChTx) or 100 nM Iberiotoxin (IbTx) in panel D. Black traces are in the absence of blockers (control) and gray traces are in the presence of each K⁺ blocker. In panel D, broken trace indicates in the presence of IbTx. Panel E shows the mean ± SE of the normalized hyperpolarization change of sperm from three different animals with triplicate measurements. Asterisks (*) indicate statistically significant differences compared to controls using Student's t test (n= 3; p ≤ 0.05). We also analyzed the data with ANOVA comparing each condition, however no significant differences between groups were found.

Zn²⁺ induces an increase in cAMP but not in cGMP

Since cyclic nucleotides play fundamental roles in the speract signaling (Darszon et al., 2008; Darszon et al., 2011; Kaupp et al., 2008), we determined the effect of Zn²⁺ on cyclic nucleotides in the cells. Speract elevates the cGMP concentration and this occurs more prominently in high K⁺ (50 mM) condition (Fig. 5A), which appears to prevent guanylyl cyclase inactivation (Harumi et al., 1992; Ward et al., 1986). In contrast, Zn²⁺ did not alter the level of cGMP either in normal K⁺ (10 mM) or high K⁺ condition (Fig. 5A). However, Zn²⁺ was able to induce an increase in the cAMP concentration, as speract does (Fig. 5B and Fig. S1). Furthermore, the increase in cAMP induced by Zn²⁺ was inhibited in high K⁺ (Fig. 5B), as in the case of speract. These results suggest that Zn²⁺ activates a K⁺ channel, possibly tetraKCNG/CNGK, without altering the cGMP concentration.

Fig. 5.

Zn²⁺ and speract raise cAMP levels but only speract increases cGMP.

Concentration of cGMP (panel A) and cAMP (panel B) in spermatozoa diluted in 0CaSW with 1 mM IBMX were measured in the presence or absence of 100 nM Speract or 10 μM Zn²⁺ (0.07 nM free Zn²⁺). Also, the same measurements were performed in high K⁺ (50 mM) condition. The mean ± SE of three experiments with sperm from different animals with triplicate measurements were normalized against the control (the resting level of cyclic nucleotides). Asterisks (*) indicate statistically significant differences compared to controls (p < 0.05).

Reducing agents partially inhibit the Zn²⁺-induced hyperpolarization

Previous work implicates cysteine residues as part of the Zn²⁺ binding site that regulates some ionic channels (Choi et al., 2001; Gore et al., 2004; Hou et al., 2010). The role of cysteines in the Zn²⁺-triggered hyperpolarization was assessed under reducing and non-reducing conditions. DTT is a strong reducing agent that also acts as a metal chelator. As shown in figures 6A&C, 5 mM DTT diminishes (~60%) the Zn²⁺-triggered hyperpolarization, but does not affect the one induced by Speract (Figs. 6B&C). As DTT is a Zn²⁺ chelator, we tested another cysteine reducing agent such as E-mercaptoethanol that is not considered a Zn²⁺ chelator. This latter reducing agent also inhibited the Zn²⁺-triggered hyperpolarization but to a lesser extent (~34%) (Fig. 6A&C). These findings suggest that cysteines do participate in this event. It is worth pointing out that the Speract induced hyperpolarization was not significantly sensitive to -mercaptoethanol or DTT. Notably, the depolarization and repolarization that follow the initial hyperpolarization induced by Zn²⁺ was differentially inhibited by DTT (~90%) and β-mercaptoethanol (~70%), while the one triggered by Speract was insensitive to both reducing agents.

Fig. 6.

The Zn²⁺-triggered hyperpolarization is inhibited by DTT and β-mercaptoethanol , two reducing agents. Sperm diluted in ASW containing or not (Control) 5 mM of DTT or β-mercaptoethanol, were exposed to either 10 μM Zn²⁺ (A) or 100 nM Speract (B). The mean ± SE of the hyperpolarization change (mV) is shown (n ≥ 5; *p ≤ 0.05).

Zn²⁺ triggers [Ca²⁺]_i fluctuations

One of the striking effects of speract on sea urchin sperm is the induction of [Ca²⁺]_i fluctuations (Wood et al., 2003). Surprisingly, we observed that Zn²⁺ induces [Ca²⁺]_i fluctuations in a similar way as speract does (Fig. 7). As expected, 50KSW completely blocked the [Ca²⁺]_i fluctuations induced by Zn²⁺ (Fig. 7) as in the case of speract (Wood et al., 2003). These results support our hypothesis, namely, that Zn²⁺ activates the tetraKCNG/CNGK channel.

Fig. 7.

Speract and Zn²⁺ induce [Ca²⁺]_i oscillations in sea urchin sperm.

Ca²⁺ images of individual spermatozoa were acquired using Fluo-4 as described in materials and methods. Panel A and B show representative fluorescence images before (A) and 3 s after addition of 100 nM speract (B). Panel C and D show representative fluorescence images before (C) and 2.5 s after addition of 10 μM Zn²⁺ (total) (D). Fluorescence images (A-D) are expressed as gray scales. Panel E, F and G indicate fluorescence changes (F/F₀) of single spermatozoon (black lines are from the head and gray ones are from the tail). Spermatozoa were stimulated by 100 nM speract (E), 10 μM Zn²⁺ (F) and 10 μM Zn²⁺ in high K⁺ ASW, 50KSW (G). The results are representative of 5 independent experiments.

High K⁺ ASW only partially inhibits Zn²⁺-induced increases in pHi and [Ca²⁺]_i

It is known that all speract-induced sperm responses, except for the cGMP increase, are suppressed in high K⁺ seawater (Harumi et al., 1992). However, we observed that Zn²⁺ only mildly inhibits an Em depolarization under these conditions (Fig. 3A). Therefore, we explored the effects of Zn²⁺ on [Ca²⁺] and pH_i changes in the presence of high external K⁺. Unexpectedly, although the Zn²⁺ induced increases in pH_i (Fig. 8A&C) and [Ca²⁺]_i (Fig. 8B&C) were smaller in 50KSW, a significant fraction of them persisted. These results suggest that there may be another target (or targets) for Zn²⁺ besides a K⁺ channel in L. pictus sperm.

Fig. 8.

High K⁺ ASW (50KSW) partially inhibits the Zn²⁺ induced increase in pH_i and [Ca²⁺]_i. Sperm pH_i and [Ca²⁺] changes induced by 10 μM Zn (1.8 nM free Zn²⁺) were measured in normal ASW (black line) and 50KSW (gray line) by fluorometry as in Figure 1. Panel A and B show representative traces of pH_i and [Ca²⁺]_I measurements, respectively. Panel C summarizes the inhibition of the pH_i and [Ca²⁺]_i increases induced by Zn²⁺ in 50KSW normalized against the values obtained in normal ASW (mean ± SE, n=3 for [Ca²⁺]_i and n=4 for pH_i).

Discussion

The tetraKCNG/CNGK channel as a principal target of Zn²⁺

It was known that Zn²⁺ is involved in pH_i regulation in sea urchin sperm, but it had not been explored whether this divalent cation affects other physiological parameters such as Em or [Ca²⁺]_i. Therefore, we first determined how Zn²⁺ affects sperm Em, pHi and [Ca²⁺]_i, and compared these responses to those induced by FSP and speract. Remarkably, our results show that Zn²⁺ also triggers sperm Em and Ca²⁺ changes besides alkalinizing pH_i (Fig. 1), as FSP and speract do. Zn²⁺ potently hyperpolarized sperm Em in a dose-dependent manner (Fig. 2). This Zn²⁺-induced hyperpolarization is likely due to the activation of K⁺ channels since 50KSW efficiently inhibited the hyperpolarization, but 0ClSW did not (Fig. 3B). However, none of the K⁺ channel blockers so far tested efficiently inhibited the Zn²⁺-induced hyperpolarization. TEA, a general K⁺ channel blocker, did not affect Zn²⁺ induced hyperpolarization. Two K_ATP channel blockers, Tolbutamide or Glibenclamide, were unable to inhibit the Zn²⁺-induced hyperpolarization although K_ATP channels are known to be activated by low Zn²⁺ concentrations (Bloc et al., 2000; Prost et al., 2004). Considering the proposed speract signaling cascade of sea urchin sperm, at least two K⁺ channels could potentially be responsible for the main component of the Zn²⁺- induced hyperpolarization; the TetraKCNG (Galindo et al., 2007) and a Ca²⁺ dependent K⁺ channel (CaKC) (reviewed in Darszon et al., 2011). The fact that the Zn²⁺-triggered hyperpolarization in L. pictus sperm was ~30 % reduced in the absence of external Ca²⁺ (Fig. S2) is, in principle, consistent with the participation of CaKCs. However, two peptide blockers of BK/Slo1, Charibdotoxin (also for Kv1 channels) and Iberiotoxin, did not significantly alter the hyperpolarization magnitude. These results may indicate the minor participation of another Ca²⁺ dependent K⁺ channel such as the small conductance SK channel (Adelman et al., 2012). Aside from this minor component, the pharmacological profile determined here coincides with the one displayed by the cGMP-gated K⁺ channel in sea urchin sperm reported by Galindo et al. previously (Galindo et al., 2000) and thereafter identified as tetraKCNG/CNGK channel (Bonigk et al., 2009; Galindo et al., 2007).

If Zn²⁺ directly activates tetraKCNG/CNGK channel, this divalent cation should stimulate the speract signaling cascade. This proposal is further supported by two different lines of experimental evidence. One is that Zn²⁺ increases cAMP levels depending on the extracellular K⁺ concentration (Fig. 5B). The other is that Zn²⁺ induces sperm [Ca²⁺]_i fluctuations as speract does (Fig. 7), in spite of not increasing cGMP levels. These findings altogether are consistent with the proposal that Zn²⁺ activates tetraKCNG/CNGK channels in sea urchin sperm. However, since there is no specific channel blocker for this channel, its heterologous expression will be required to establish if Zn²⁺ directly activates tetraKCNG/CNGK or not.

Cysteine residues have been found as part of the Zn²⁺ binding site that regulates several membrane proteins including ionic channels (Choi et al., 2001; Gore et al., 2004). Our experiments showed that neither DTT nor -mercaptoethanol, two reducing agents, decreased the hyperpolarization triggered by Speract (Fig. 6B&C), however, both compounds inhibited the Zn²⁺- induced hyperpolarization (Fig. 6A&C; ~60 % and ~35 %, respectively). It is important to point out that the TetraKCNG contains three extracellular cysteines (C214 in IS5-S6; C1311 in IIIS5-S6 and C1946 in IVS5-S6) one of which could be a Zn²⁺ target. Our findings thus suggest that the K⁺ channel activated by Zn²⁺ has a cysteine binding site and if it is tetraKCNG, the Zn²⁺ binding site and the one for speract are independent. Certainly it is possible that a sea urchin sperm K⁺ channel still not described could be the main target of Zn²⁺.

Other targets of Zn²⁺ besides tetraKCNG/CNGK channel

Although the tetraKCNG/CNGK channel is likely to be the principal target of Zn²⁺ in the sea urchin sperm plasma membrane, it is probably not the only one. High [K⁺]_e inhibited only ~50 % of the Zn²⁺-triggered Ca²⁺ increase (Fig. 4C&D) and ~40 % of the pHi alkalinization. This divalent cation could activate directly or indirectly Ca²⁺ channels besides K⁺ channels and the sNHE. It is important to stress that the Zn²⁺ induced increase in pHi does not by itself cause a hyperpolarization ((Garcia-Soto et al., 1987) and experiments not shown). Furthermore, Zn²⁺ could affect a carbonic anhydrase found in the S. purpuratus genome (similar to carbonic anhydrase 8, NCBI Reference Sequence: XP_795365.2). It is therefore clear that Zn²⁺ has multiple targets in sea urchin sperm as it does in many other cell types.

In high K⁺ seawater the speract signaling cascade is completely blocked, except for the cGMP elevation (Harumi et al., 1992). Namely, both the pH_i and [Ca²⁺]_i increases induced by speract are completely suppressed in this condition. In contrast, those induced by Zn²⁺ were only significantly reduced (60 and 40 %, respectively), but not eliminated. Actually, this difference may distinguish the action of Zn²⁺ and speract on L. pictus spermatozoa where Zn²⁺ is able to trigger a significantly higher % of AR than speract (Fig. S3A) as previously reported (Clapper et al., 1985). In this regard we examined if Zn²⁺ triggers the AR through the same mechanism as FSP does.

Interestingly, while Ca²⁺ in seawater is essential for the FSP-induced AR, it is not for the Zn²⁺-induced AR (Fig. S3B). Furthermore, though Ca²⁺ channel blockers, Ni²⁺ (300 μM) and nifedipine (20 μM) efficiently inhibit the AR induced by FSP, neither treatments inhibited the Zn²⁺- induced AR (Fig. S3B). Nifulmic acid, a Cl^- channel blocker, only inhibited the AR induced by FSP, but not the AR induced by Zn²⁺ (Fig. S3B). Although 50KSW efficiently inhibited the AR in both cases, the K⁺ channels involved in these two events seems different since TEA inhibits FSP-induced AR in S. purpuratus sea urchin sperm (Guerrero and Darszon, 1989), in contrast this blocker did not inhibit the Zn²⁺-induced AR. This pharmacological profile coincides with that of the Zn²⁺-induced hyperpolarization (Fig. 4). These findings altogether indicate that Zn²⁺ and FSP trigger the AR through different signaling pathways. It is worth pointing out that in S. purpuratus spermatozoa, Zn²⁺ does not induce the AR although it causes Em hyperpolarization and increases in pH_i and [Ca²⁺]_i (data not shown). The differential ability of Zn²⁺ to induce AR in the two species probably stems from the fact that L. pictus sperm have a lower threshold to trigger this reaction.

Physiological role of Zn²⁺ for sea urchin sperm

It is known that Zn²⁺ plays crucial roles for the initiation of sperm motility and the AR in L. pictus sea urchins. In both cases, pH_i regulation might be a key point. It is known that sperm motility is suppressed in the testis by low pH_i principally produced by a high partial pressure of CO₂ (Johnson et al., 1983). Upon spawning, spermatozoa are exposed to seawater that has low partial pressure of CO₂ and high pH (around 8.0), which results in the elevation of pH_i.

It was reported that a Na⁺/H⁺ exchanger (NHE) is also involved in the alkalinization of the pH_i upon sperm motility initiation (Lee et al., 1983). It has been known that sea urchin sperm possess a unique NHE which is regulated by membrane potential (hyperpolarization) (Lee, 1984; Lee and Garbers, 1986). Now, this NHE is thought to be a sea urchin homolog of sperm specific NHE (sNHE), initially identified in mouse (Nomura and Vacquier, 2006; Wang et al., 2003). Compared to other NHEs in somatic cells, this sNHE contains an extra domain composed of 4 trans-membrane segments in the C-terminus (Wang et al., 2003), which is structurally similar to the voltage sensor domain of voltage-gated channels and voltage-regulated phosphatases (Okamura, 2007). Therefore, it is believed that sNHE is regulated by membrane potential using this putative voltage sensor domain. In the speract signaling cascade, Em hyperpolarization caused by K⁺ efflux through tetraKCNG/CNGK channel is supposed to activate the sHNE and lead to pH_i alkalinization (Darszon et al., 2008). Results obtained in this study suggest that the same signaling cascade can be activated by Zn²⁺ upon sperm dilution when seawater contains Zn²⁺. It is known that low pH seawater can suppress sperm motility (Ohtake, 1976), but the sperm flagella still slightly vibrate in this condition. Related to this fact, low pH (6.0) and high K⁺ (50 mM) seawater has been employed as a condition to store immotile spermatozoa for long period (Bracho et al., 1997). Taking all information into account, the Em hyperpolarization caused by Zn²⁺-induced K⁺ efflux might be relevant for sperm motility initiation upon spawning in seawater. It is worth mentioning that, in the ascidian Ciona intestinalis, an egg-derived single ligand, a sulfated steroid named SAAF, induces both sperm motility initiation and sperm chemotaxis (Yoshida et al., 1993; Yoshida et al., 2002). SAAF is known to hyperpolarize sperm Em through K⁺ efflux (Izumi et al., 1999). Furthermore, it was reported, in gill cells of the mollusk Mytilus galloprovincialis, that Zn²⁺ activates a NHE and increases pH_i (Kaloyianni et al., 2006; Koutsogiannaki et al., 2006). Since we observed that Zn²⁺ still increases sperm pH_i in high K⁺ seawater (Fig. 8), it cannot be ruled out the possibility that this divalent cation directly activates the sNHE.

Conclusions

We demonstrated that submicromolar free Zn²⁺ in seawater can activate a K⁺ channel of sea urchin spermatozoa. Pharmacological profiles suggest that the tetraKCNG/CNGK channel, which plays the fundamental role in the early part of the speract signaling cascade, is a principal target of Zn²⁺. Therefore, Zn²⁺ can promote sperm motility initiation upon spawning by sharing actions of speract: increases in pH_i , [Ca²⁺]_i and cAMP. Further detailed studies, particularly heterologous expression of tetraKCNG/CNGK channel, are necessary to define the molecular action of Zn²⁺ in sea urchin spermatozoa.

Zn²⁺ hyperpolarizes sea urchin sperm membrane potential.

The cGMP-gated K⁺ channel seems to be a main target of Zn²⁺.

Zn²⁺ induces [Ca²⁺]_i fluctuations.

Zn²⁺ increases cAMP, but not cGMP.