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Biol Reprod. 2009 February; 80(2): 311–319.
Prepublished online 2008 October 15. doi:  10.1095/biolreprod.108.071076
PMCID: PMC2804820

Egg Water from the Amphibian Bufo arenarum Modulates the Ability of Homologous Sperm to Undergo the Acrosome Reaction in the Presence of the Vitelline Envelope1

Abstract

Sperm from the toad Bufo arenarum must penetrate the egg jelly before reaching the vitelline envelope (VE), where the acrosome reaction is triggered. When the jelly coat is removed, sperm still bind to the VE, but acrosomal exocytosis is not promoted. Our previous work demonstrated that diffusible substances of the jelly coat, termed “egg water” (EW), triggered capacitation-like changes in B. arenarum sperm, promoting the acquisition of a transient fertilizing capacity. In the present work, we correlated this fertilizing capacity with the ability of the sperm to undergo the acrosome reaction, further substantiating the role of the jelly coat in fertilization. When sperm were exposed to the VE, only those preincubated in EW for 5 or 8 min underwent an increase in the intracellular Ca2+ concentration ([Ca2+]i), which led to acrosomal exocytosis. Responsiveness to the VE was not acquired on preincubation in EW for 2 or 15 min or in Ringer solution regardless of the preincubation time. In contrast, depletion of intracellular Ca2+ stores (induced by thapsigargin) promoted [Ca2+]i rise and the acrosome reaction even in sperm that were not exposed to EW. Acrosomal exocytosis was blocked by the presence of Ca2+ chelators independent of whether a physiological or pharmacological stimulus was used. However, Ni2+ and mibefradil prevented [Ca2+]i rise and the acrosome reaction of sperm exposed to the VE but not of sperm exposed to thapsigargin. These data suggest that the acrosomal responsiveness of B. arenarum sperm, present during a narrow period, is acquired during EW incubation and involves the modulation of a voltage-dependent Ca2+ channel.

Keywords: acrosome reaction, amphibia, capacitation, fertilization, jelly coat, spermatozoa, sperm capacitation

INTRODUCTION

Mature oocytes of most animal species are surrounded by extracellular matrices. These vestments, structurally and morphologically distinct among species, are the first barrier that sperm must pass through before reaching the egg plasma membrane. Various investigations have assigned multiple functions to these barriers such as species selectivity, induction of the acrosome reaction, and prevention of polyspermy. In amphibians, the extracellular matrix is formed by the vitelline envelope (VE [analogous to the zona pellucida in mammals]) and by the surrounding jelly coat deposited during the oocyte transit through the oviduct. The passage of sperm through the jelly coat has been regarded as an important step in fertilization and was sometimes proposed to be a sperm “capacitating” requisite by analogy with the concept developed in mammals [1]. Dejellied oocytes of different amphibian species can be fertilized after reintroduction of the diffusible jelly components, termed “egg water” (EW), in the insemination media [2, 3]. The EW was reported to “activate” free spermatozoa of the toad Bufo arenarum and to maintain acrosome integrity, preventing hypo-osmotic shock before they penetrate into the jelly coats [48]. Our previous work demonstrated that incubation in EW for 8 min was sufficient to render B. arenarum sperm transiently capable of fertilizing dejellied oocytes [9]. The fertilizing state was correlated with an increase in protein tyrosine phosphorylation and a decrease in sperm cholesterol content. These changes are reminiscent of mammalian sperm capacitation and take place before the acrosome reaction [9].

The acrosome reaction in the toad [10] is similar to that in mammals [11] because it comprises exposure of the inner acrosomal membrane without formation of a prominent acrosomal process. A necessary condition for B. arenarum spermatozoa to fertilize the oocyte is to reach the VE with the acrosome intact [10, 12, 13]. Previous work showed that the acrosome of Bufo japonicus sperm bound to the VE of dejellied oocytes is not reacted [12]. Because the occurrence of the acrosome reaction is an absolute prerequisite for fertilization in all species with an acrosome, sperm that fail to undergo the acrosome reaction are denied access to the oocyte membrane.

In this article, we provide evidence indicating that triggering of physiological acrosomal exocytosis in B. arenarum sperm depends on an incubation period in EW. This incubation promotes a transient capacitated state in sperm that enables it to undergo a rise in intracellular Ca2+ concentration ([Ca2+]i) in response to the VE, leading to an acrosome reaction. Pharmacological increase in [Ca2+]i due to the release of Ca2+ from intracellular stores on exposure of sperm to thapsigargin promoted an acrosome reaction independent of the presence of EW. Our results also show that the VE- and thapsigargin-induced acrosome reaction is blocked by the presence of Ca2+ chelators in the extracellular medium. Ca2+ mobilization during the onset of the acrosome reaction is discussed.

MATERIALS AND METHODS

Reagents

Thapsigargin was purchased from Calbiochem (La Jolla, CA). Fluo3-AM (a fluo3 ester form) was obtained from Biotium, Inc. (Hayward, CA) and was prepared as a 5 mM stock solution in dimethyl sulfoxide; aliquots were stored at −20°C. Mibefradil dihydrochloride was obtained from Sigma (St. Louis, MO), dissolved in distilled water, and aliquoted into individual vials stored at −20°C until required. Secondary mouse anti-rabbit antibody, conjugated to Cy3, was purchased from Chemicon (Temecula, CA). All other chemicals were of reagent grade.

Animals

Bufo arenarum sexually mature specimens (150 g) were collected in the neighborhood of Rosario, Argentina, and maintained in the dark in a moist chamber between 15°C and 17°C until used. Experiments were performed in accord with the guide for the care and use of laboratory animals of Facultad de Ciencias Biológicas y Farmacéuticas, Universidad Nacional de Rosario.

Preparation of Gametes

Sperm suspensions were obtained as described elsewhere [14]. After washing, spermatozoa were suspended in ice-cold Ringer solution (110 mM NaCl, 2 mM KCl, 1.4 mM CaCl2, 10 mM Tris-HCl [pH 7.6]) to a final concentration of 1–1.4 × 108 cells/ml and were used within 3 h. The VE was obtained from oocytes collected form the ovisac (referred to as oocytes) as described [15]. Recovered VE was rinsed twice with Ca2+-free Ringer solution and finally diluted three times with distilled water. The VE samples used throughout this work were sonicated 3 × 30 sec at 80 W [16] to avoid heat denaturation that would result in loss of the VE ultrastructure. Standardization of samples was performed measuring total proteins of solubilized VE fractions (10 min at 100°C) of different preparations. Samples were adjusted to 0.8 μg/μl of total proteins.

Egg Water

The EW was obtained as described [17] and supplemented with 0.1 volume of 10× Ringer solution to obtain isotonic solutions. The resulting tonicity blocks sperm motility induction [18], preventing measurement interference by intracellular Ca2+ modifications related to flagella activation. Total protein concentration in 80% EW-Ringer solution was 0.24 mg/ml. This solution is referred to as EW.

Analysis of the Acrosome Reaction

Sperm (5 × 106 cells/ml) were incubated in 500 μl of EW or Ringer solution supplemented with 10 mM EDTA, 10 mM EGTA, or variable concentrations of Ni2+ or mibefradil as indicated. Sonicated VE (final concentration, 6.4 μg/ml) was added after the specified periods. Alternatively, the effect of thapsigargin on the acrosome reaction was analyzed. Ten microliters of sperm suspensions were fixed with 1.5% formaldehyde after the indicated periods and smeared onto a slide to evaluate acrosome status of the spermatozoa. Intact acrosomes were detected in unreacted sperm as previously described [19]. Briefly, acrosomes were immunostained using rabbit anti-acrosomal matrix antibodies and a Cy3-conjugated secondary antibody. This maneuver allows detection and identification of reacted and unreacted sperm by acrosome staining, located in the sperm apical tip (See Figure 2, A and B). Samples were examined with phase-contrast and epifluorescence microscopy using an Olympus (Tokyo, Japan) BH2 microscope equipped with a mercury short-arch lamp. Micrographs were taken with a Nikon (Natick, MA) DS-Fi1 camera and a DS-U2 control unit. At least 150 spermatozoa were counted on each slide.

Loading of Fluorescent Indicator

Sperm cells (7.2 × 107 sperm/ml) were incubated in the dark at 18°C for 90 min in 1 ml of Ringer solution containing 3 μM fluo3-AM. To remove excess dye, cells were washed once and resuspended at original concentration in Ringer solution. Suspensions were maintained at 18°C for 30 min to allow the intracellular liberation of the indicator. Sperm were washed and resuspended at original concentration (7.2 × 107 sperm/ml) and kept on ice and used within 3 h. Microscopic examination of sperm loaded with fluo3 (in Ringer solution) showed uniform low fluorescence, indicating that the fluorophore was distributed throughout the cytosolic space.

Fluorescence in Cell Suspensions

The fluorescence signal was measured at 20°C under continuous stirring in a Varian (Palo Alto, CA) Cary Eclipse spectrofluorometer. The experimental procedure was initiated by adding Ringer solution or EW to the cuvette. Trace recording began immediately before diluting the dye-loaded cell suspension in the cuvette. This provided 3 × 106 sperm/ml. Samples were excited at 495 nm, and the emitted light was selected at 530 nm (5-nm band pass), with automatic filter adjustment at 0.85 Hz. Fluo3 fluorescence of loaded sperm was used to study intracellular free Ca2+ variations. The maximum fluorescence (fmax) was that observed on addition of 0.1% Triton X-100 to the sperm suspension; the minimum fluorescence (fmin) was that observed on subsequent addition of 10 mM EDTA. This maneuver performed for each sample allowed normalization of cytoplasmic free Ca2+ variations as a fraction of the difference between fmax and fmin (relative arbitrary fluorescence units [AFUs]), rather than as AFUs, which are far more variable. Therefore, relative AFUs were calculated as [F (fmax of the peak observed) − F0 (fluorescence previous to the stimulus)]/(fmaxfmin), as shown in Figure 1, C and D. Fluorescence arising from the external medium due to the passive release of fluo3 was negligible when cells were diluted in the absence of Ca2+ (i.e., when Ringer solution with EDTA was the dilution medium). The traces shown in the figures are representative of three to five independent experiments. The software for smoothing the experimental traces used the Savitsky-Golay algorithm.

FIG. 1.
[Ca2+]i increase driven by the VE in sperm incubated in deferent medium. A) Sperm were loaded with fluo3 and incubated in EW or Ringer solution. Sonicated VE was added (as indicated by the arrowheads) after the incubation period specified. Traces are ...

Statistical Analysis

Statistical analyses were performed using ANOVA. Models were further tested according to Nagarsenker [20] and Shapiro and Wilk [21]. Significance (P) and sample size (n) are indicated in each figure legend.

RESULTS

VE Regulates Sperm [Ca2+]i

A common feature in the regulation of exocytotic processes is the general role of [Ca2+]i as a mediator of stimulus-secretion coupling. In sperm and somatic cells, increases of [Ca2+]i are necessary but not always sufficient to initiate the secretion [11, 2224]. Extracellular Ca2+ seems to be necessary for the completion of mammalian sperm capacitation in vitro [25] when an increase in [Ca2+]i has been demonstrated [26]. To analyze whether [Ca2+]i changes were related to the acquisition of fertilizing capacity of B. arenarum spermatozoa, sperm were loaded with fluo3-AM and then incubated in EW for 30 min. [Ca2+]i was continuously analyzed during this incubation period. Although sperm incubation in EW has been shown to promote fertilization [9], [Ca2+]i changes could not be detected (data not shown), suggesting that [Ca2+]i alteration is not involved in the acquisition of fertilizing capacity. However, a significant increase in intracellular Ca2+ was observed after sperm exposure to the VE. Sperm were incubated for various periods in EW before addition of the VE. [Ca2+]i rise was observed as a fluorescence intensity increase (Fig. 1, A and C). The addition of VE (6.4 μg/ml of final protein concentration) resulted in a steep transient rise in [Ca2+]i (Fig. 1, A and D). This response seemed to be dependent on the sperm incubation period in EW. The transient rise in [Ca2+]i could only be detected when sperm were preincubated for 5 or 8 min in EW. This peak was followed by a decrease in [Ca2+]i that did not reach basal levels and was sustained for at least 5 min more when the measurement was stopped (Fig. 1, A and C). However, the increase in sperm [Ca2+]i was significantly reduced when the VE was added to sperm incubated in EW for 2 or 15 min and was absent in sperm incubated in control Ringer solution without the addition of EW. These results indicate that B. arenarum sperm need to be in the presence of EW for a discrete period to be able to undergo the acrosome reaction when the VE is added. This time window in the presence of EW is similar to that observed in fertilization assays of dejellied eggs [9].

Influx of Ca2+ across the plasma membrane is a common mechanism of [Ca2+]i signaling. To study whether extracellular Ca2+ is involved in the VE-induced transient rise in [Ca2+]i, we incubated sperm in the presence of Ca2+ chelators. As calculated using MaxChelator software Webmaxc Standard (http://www.stanford.edu/~cpatton/webmaxcS.htm) [27], when 10 mM EDTA and EGTA was added to EW, free Ca2+ concentration was about 4 nM and 6 nM, respectively. The VE was added to the sperm suspension after 8 min of incubation in free Ca2+-EW, a period sufficient to enable a sperm Ca2+ response to the VE (Fig. 1A). In this case, no VE-driven Ca2+ influx was noted, indicating that extracellular Ca2+ chelators blocked the increase in [Ca2+]i (Fig. 1B). Each individual trace was calibrated as described in Materials and Methods (Fig. 1C), and these data were used for quantification (Fig. 1D).

Sperm Incubation in the Presence of EW Is Needed for the Physiologically Induced Acrosome Reaction

The cytoplasmic [Ca2+]i rise induced by stimulation with the VE was observed only in sperm samples preincubated in EW for 5 or 8 min. As already mentioned, these periods are correlated with the fertilization-supporting window displayed by B. arenarum sperm incubated in EW [9]. We hypothesized that sperm acquire the ability to trigger the acrosome reaction on VE stimulation only after these exposures. To test this hypothesis, the kinetics of the acrosome reaction in B. arenarum sperm was first investigated. Sperm suspensions were preincubated in EW for 5 min before exposure to the VE to allow the acquisition of fertilizing capacity [9]. Samples were fixed after 1.5, 4, and 10 min of exposure to the VE, and the acrosomal status was evaluated (Fig. 2, A and B). The VE-induced acrosome reaction had peaked by 1.5 min, the shortest period tested (Fig. 2C). These data indicate that the acrosome reaction in capacitated sperm is a fast process that takes place during the first 90 sec after physiological stimulation. To address the effect of the EW preincubation time on the VE-induced acrosome reaction, sperm suspensions were incubated for variable periods in EW or Ringer solution before addition of the VE and subsequently fixed after 90 sec. High rates of acrosome reaction were observed after 5 and 8 min of sperm preincubation in EW (Fig. 2D). The acrosome reaction was not triggered after preincubation in Ringer solution. Moreover, the VE-induced acrosomal exocytosis was significantly reduced when B. arenarum sperm were incubated in EW for 2 or 15 min (Fig. 2D). These data suggest that the transient fertilizing capacity of B. arenarum sperm as observed by in vitro fertilization assays [9] can be, at least in part, ascribed to a transient capability to undergo the acrosome reaction after the VE stimulus.

FIG. 2.
The acrosome reaction in B. arenarum sperm triggered by the VE. A and B) Immunocytochemical analysis of acrosomal status in B. arenarum spermatozoa. Acrosomes were immunodetected with anti-acrosomal matrix antibodies and stained with Cy3-conjugated secondary ...

Role of Intracellular Ca2+ Stores in the Acrosome Reaction of B. arenarum Sperm

In somatic cell types, thapsigargin induces an inositol 1,4,5-triphosphate-independent [Ca2+]i increase; this effect is mediated by the inhibition of Ca2+-ATPase pumps localized in the membrane of internal Ca2+ stores [28]. The acrosomal vesicle of human sperm was identified as an internal Ca2+ store sensitive to thapsigargin, which is involved in the [Ca2+]i rise observed after zona pellucida stimulation [24, 29, 30]. To assess whether intracellular Ca2+ stores in B. arenarum sperm are involved in the events leading to the acrosome reaction, Ca2+ mobilizations that could result from thapsigargin exposure were evaluated. Addition of this drug to sperm suspended in Ringer solution induced a concentration-dependent rise in [Ca2+]i (Fig. 3A). Maximum [Ca2+]i increase was observed after treatment with 5 μM thapsigargin. No further increase was observed with 10 μM thapsigargin (data not shown). The presence of EDTA or EGTA in the medium did not significantly reduce the Ca2+ response to thapsigargin (Fig. 3, A and B). However, when 1 μM or 5 μM thapsigargin was added to sperm in Ca2+-free Ringer solution, [Ca2+]i returned to lower basal levels than those observed in Ringer solution.

FIG. 3.
Increase in [Ca2+]i in B. arenarum sperm promoted by thapsigargin (Thap). Fluo3-loaded sperm were suspended in Ringer solution or EW supplemented with EDTA or EGTA (10 mM) as specified. A) Ringer solution-incubated sperm were exposed to variable concentrations ...

Different laboratories have shown that the increase in [Ca2+]i induced by thapsigargin in capacitated mammalian sperm leads to the acrosome reaction [31, 32], probably through capacitative Ca2+ entry [33, 34]. To investigate the acrosomal responsiveness of B. arenarum sperm to thapsigargin, the acrosomal status was evaluated after exposing sperm to different concentrations of the drug. Thapsigargin was able to induce the acrosome reaction in a concentration-dependent manner (Fig. 3C). Sperm treatment with 1 μM and 5 μM thapsigargin produced a significant increase in acrosome reaction percentages (P < 0.05). However, this effect was abrogated when EDTA or EGTA was present in the medium. Furthermore, 5 μM thapsigargin in the presence of EDTA or EGTA induced a [Ca2+]i rise even higher than that promoted by 1 μM thapsigargin in Ringer solution-incubated sperm or by the VE in capacitated sperm. However, the acrosome reaction was prevented, indicating that the presence of chelators in the extracellular medium prevented acrosomal exocytosis.

Because sperm were no longer able to respond to the VE after 15 min of incubation in EW, it could be hypothesized that this solution damages sperm, rendering cells unable to undergo acrosomal exocytosis. However, thapsigargin was still capable of promoting [Ca2+]i rise and the acrosome reaction when challenged after a preincubation of 15 min in EW (Fig. 3, D and E).

Ca2+ Mobilization Triggered by the VE

Ca2+ uptake that occurs during the acrosome reaction in mammalian and sea urchin sperm, as well as Ca2+ mobilization itself, is inhibited by dihydropyridines, diphenylalkylamines, and Ni2+ [3537]. These data suggested the participation of voltage-dependent Ca2+ channels during these processes. Although nifedipine and related dihydropyridines were considered initially as specific L-type homovanillic acid channel antagonists, these compounds also were found to block T-type currents when used at micromolar concentrations [3840]. T-type Ca2+ currents have low voltage activation thresholds and are inhibited by mibefradil and Ni2+ at micromolar concentrations. In light of these data, the effect of mibefradil and Ni2+ on Ca2+ mobilization of B. arenarum sperm was assessed. Cells were incubated in EW-Ni2+ or EW-mibefradil for 8 min before exposure to the VE. The rise in [Ca2+]i previously observed on physiological stimulation was blocked in a concentration-dependent manner (Fig. 4, A and B). Both inhibitors displayed partial inhibition at the lowest concentration tested (1 μM). At 50 and 250 μM Ni2+ addition, the internal Ca2+ increase was clearly abolished. When 5 μM mibefradil was present in the medium, Ca2+ mobilization was completely abrogated. Neither Ni2+ nor mibefradil blocked [Ca2+]i rise when sperm were subsequently treated with 5 μM thapsigargin (Fig. 4C). Moreover, as already mentioned, thapsigargin induced a transient peak in [Ca2+]i that is then slowly reduced to a steady state that is much higher than the basal level. When the experiment was conducted in the presence of EDTA or EGTA, the [Ca2+]i steady state was significantly reduced and reached basal levels (Fig. 3A). In contrast to extracellular Ca2+ chelators, Ni2+ and mibefradil did not reduce the thapsigargin-associated [Ca2+]i steady-state level (Fig. 4C). These data suggest that the Ca2+ channels blocked by this inhibitor are not the ones needed to maintain high [Ca2+]i after thapsigargin induction.

FIG. 4.
Effect of Ca2+ channel antagonists on [Ca2+]i. A) Fluo3-loaded sperm were incubated in EW supplemented with different amounts of Ni2+ or mibefradil (mib) for 8 min. The VE was added as indicated by the arrowheads. Traces are representative of three independent ...

To evaluate the effect of Ca2+ mobilization blockade on the acrosome reaction, sperm were incubated in EW supplemented with Ni2+ or mibefradil before stimulation with the VE or thapsigargin. Consistent with the inhibitory effect of Ni2+ and mibefradil on [Ca2+]i, when B. arenarum sperm were challenged with the VE, the presence of these inhibitors blocked the acrosome reaction in a concentration-dependent manner (Fig. 5A). In contrast, Ni2+ and mibefradil did not inhibit the thapsigargin-induced acrosome reaction (Fig. 5B). These data are in accord with the lack of effect of these Ca2+ channel blockers on the thapsigargin-stimulated [Ca2+]i increase.

FIG. 5.
Effect of Ca2+ channel antagonists on the acrosome reaction. Sperm were incubated for 8 min in EW supplemented with Ni2+ or mibefradil (mib) before exposure to the VE (A) or to thapsigargin (Thap) (B). Cells were fixed and processed for acrosomal status ...

DISCUSSION

Our previous study [9] demonstrated that B. arenarum sperm should undergo a series of molecular changes to gain fertilizing capacity. The fertilizing state had been correlated with an increase in protein tyrosine phosphorylation and a decrease in sperm cholesterol content. These changes promoted by incubation in EW take place before the acrosome reaction. It is well known that the water-soluble components of the jelly coat support fertilization of jellyless oocytes in a number of amphibian species [5, 41, 42]. However, the question of how these factors affect sperm-fertilizing capacity has remained poorly understood.

A previous study [43] had paid special attention to the ionic components of the jelly coat and concluded that Ca2+ is an essential constituent of EW. Fertilization of dejellied oocytes was achieved in a salt solution containing Ca2+ and was impaired by EDTA addition. These experiments were performed in the presence of HCo3 in the insemination media. We have shown that HCo3 can substitute for EW in supporting acquisition of sperm-fertilizing capacity [9]. These observations suggest that Ca2+ may not be the only requirement for fertilization of dejellied oocytes.

Ca2+ is an essential mediator of the acrosome reaction in all species studied [38, 44]. In the present work, we studied the regulation of sperm [Ca2+]i during fertilization. The VE of B. arenarum oocytes drove a rapid rise in sperm [Ca2+]i, followed by a decrease, similar to what was observed by Bailey and Storey [45] in mouse sperm. The dwindling from maximal to steady state [Ca2+]i occurred with half-time of 15 sec. When extracellular Ca2+ was lowered to the nanomolar range by addition of Ca2+ chelators, Ca2+ mobilization was impaired, pointing to the importance of extracellular Ca2+ in this process. The transient peak of [Ca2+]i was correlated with the triggering of the acrosome reaction: when a transient rise in [Ca2+]i was detected after VE stimulation, sperm underwent acrosomal exocytosis. Bufo arenarum sperm acquired the capacity to undergo the acrosome reaction after 5 or 8 min of incubation in EW. However, after a 15-min period of incubation in EW, the ability of sperm to undergo the acrosome reaction on exposure to the VE was severely diminished. Sperm remained capable of undergoing acrosomal exocytosis when challenged with thapsigargin after 15 min of incubation in EW. These data suggest that the capacity of sperm to undergo the VE-induced acrosome reaction is present only during a few minutes and only once in the lifetime of the sperm. Our previous study [9] showed a sudden decrease in fertilizing capacity when sperm preincubated in EW longer than 8 min were used for inseminating jellyless oocytes. This observation could now be explained, at least in part, by the lack of sperm capacity to trigger acrosomal exocytosis. The acrosome reaction was observed only in sperm that were incubated in EW but not when incubated in Ringer solution, further substantiating the role of jelly components in fertilization. The EW and Ringer solution have similar Ca2+ concentrations (approximately 1.5 mM). However, sperm were capable of undergoing acrosomal exocytosis only when incubated in EW, indicating that Ca2+ is necessary in the inseminating media but is insufficient for sperm to undergo the acrosome reaction and fertilize.

In B. arenarum sperm, thapsigargin induced a rapid [Ca2+]i rise in sperm suspended in Ringer solution in the presence or absence of available extracellular Ca2+; however, when extracellular EDTA or EGTA was added, the acrosome reaction was blocked. Chelators could affect Ca2+ requirements for membrane fusogens [46]. As a likely possibility, Ca2+ influx through store-operated Ca2+ channels might also be involved, as suggested for mammalian sperm acrosome reaction [24, 47]. Thapsigargin, an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPase, also induces cytoplasmic Ca2+ rise in mammalian sperm [31, 32]. This increase is likely to be due to Ca2+ depletion from internal stores caused by thapsigargin, which leads to a sustained influx of extracellular Ca2+ through store-operated channels [48]. Consistent with this hypothesis, antibody inhibition of a transient receptor potential channel in mouse sperm has been shown to block the thapsigargin-activated Ca2+ entry pathway that drives the acrosome reaction [34]. In B. arenarum sperm, [Ca2+]i rise and the acrosome reaction induced by thapsigargin were insensitive to Ni2+ and mibefradil at concentrations up to 1 mM and 5 μM, respectively. A concentration of 1 mM Ni2+ did not effectively block store-operated Ca2+ influx involved in ascidian sperm acrosome reaction [49].

The following two main differences are noted when comparing thapsigargin-promoted responses in B. arenarum sperm with observations in sea urchin counterparts: 1) thapsigargin does not cause a substantial increase in [Ca2+]i in sea urchin sperm when Ca2+ is not available in the extracellular medium and 2) thapsigargin by itself does not trigger the acrosome reaction even in the presence of extracellular Ca2+ [44]. A possible explanation for these differences could be a larger intracellular Ca2+ store in B. arenarum sperm than in sea urchin, which could give a stronger Ca2+ signal. A second coordinated physiologic modification also could be required in sea urchin sperm to undergo the acrosome reaction [44]. In the case of mammalian sperm, thapsigargin was reported to have greater effect on stimulation of [Ca2+]i rise and the acrosome reaction in capacitated spermatozoa vs. noncapacitated spermatozoa [50, 51]. It has been suggested that these species require a capacitation period to fulfill intracellular reservoirs, ensuring enough Ca2+ stored to undergo the acrosome reaction [26]. This recruitment of Ca2+ might be absent in B. arenarum sperm because [Ca2+]i rise induced by thapsigargin occurred in a similar fashion when the cells were preincubated in the presence or absence of EW (Fig. 3, A and D). However, Ca2+ recruitment could have occurred during sperm preparation or fluo3 loading in Ringer solution before EW incubation.

The influx of Ca2+ into mouse and sea urchin sperm induced by the zona pelucida and the VE involves the sequential opening of two different Ca2+ channels [38, 44]. Several lines of evidence suggest that the first Ca2+ entry through voltage-sensitive channels is responsible for mediating zona pelucida signal transduction [52]. This first rapid influx (lasting 200 millisec) is believed to be coupled to inositol 1,4,5-triphosphate production, which in turn depletes intracellular sperm Ca2+ stores. As previously discussed, the presence of Ni2+ or mibefradil in Ringer solution did not modify the [Ca2+]i response when B. arenarum sperm were treated with thapsigargin. However, 1 μM Ni2+ or mibefradil (known to affect T-type Ca2+ channels) significantly decreased the VE-induced [Ca2+]i rise in sperm incubated in EW. This inhibition was correlated with the impairment of the acrosome reaction. Accordingly, the acrosome reaction of human sperm is inhibited by micromolar concentrations of Ni2+ and mibefradil [5355]. Nickel blocks Cav3.2 (a T-subtype channel) Ca2+ currents approximately ten times more potently than other Ca2+ currents. It has been reported that recombinant Cav3.2 currents are selectively blocked by low micromolar concentrations of NiCl2 [56]. Similar Ni2+ sensitivity was found to affect Cav3.2 channels from rat aorta smooth muscle cells (half-maximal blocking concentration [IC50], 10 μM) [57], rat amygdala (IC50, 30 μM) [58], and medullar thyroid carcinoma cells (IC50, 5 μM) [59]. It has been suggested that the voltage-gated channels CATSPER3 and CATSPER4 could participate in the acrosome reaction of mouse sperm [60]. However, data concerning their electrophysiology or pharmacology are not completely available, to our knowledge. Because very low concentrations of Ni2+ and mibefradil completely blocked Ca2+ mobilization and the acrosome reaction in B. arenarum sperm, a voltage-dependent Ca2+ channel could be involved in the elicited [Ca2+]i increase in an early, if not the initial, reaction in the sequence leading to physiologically induced acrosomal exocytosis.

In light of these data, it is impossible to ascertain whether thapsigargin and the VE share a common pathway that ends with the acrosome reaction. The working model for the control of B. arenarum sperm physiology during fertilization would include the modulation of sperm response to the VE that takes place during EW incubation (Fig. 6). Sperm binding to the VE does not depend on jelly coat factors [61]. However, binding of sperm to jellyless eggs in medium devoid of EW does not trigger acrosomal exocytosis. It is likely that a voltage-dependent Ca2+ channel acts as a key element of the VE signal transduction. The activity of this channel could be subjected to regulation during acquisition of fertilizing capacity. Once in an active form, the opening of this channel due to the VE binding would lead to [Ca2+]i rise. Downstream of this point, three different possible mechanisms arise: 1) this intracellular Ca2+ increase is sufficient to trigger the acrosome reaction; 2) the initial Ca2+ influx leads to intracellular Ca2+ store depletion, triggering the acrosome reaction; or 3) depletion of intracellular stores would lead to a capacitative Ca2+ entry, promoting acrosomal exocytosis. The capacity of sperm to respond to the VE is present only during a few minutes. Understanding EW-induced signaling events, as well as the function of the jelly components, in supporting fertilizing capacity of B. arenarum sperm warrants further investigation.

FIG. 6.
Working model of cellular changes elicited on B. arenarum sperm capacitation and exposure to the VE, with subsequent triggering of the acrosome reaction. The left half of the scheme involves previous data concerning B. arenarum sperm acquisition of fertilizing ...

Acknowledgments

We thank M. Leiva, L. Racca, and H. Bottai for statistical analyses.

Footnotes

1Supported by funding from the Josefina Prats Foundation to D.K., by grants PICT010-8545 and PICT15-31660 from the Agencia Nacional de Promoción Científica y Tecnológica and grant PIP6428 from CONICET to M.O.C. and S.E.A., and by grants HD38082 and HD44044 from the National Institutes of Health to P.E.V.

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