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Biology of Reproduction 63, 635-642 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular Article, PCC

Gonadotropin-Releasing Hormone-Stimulated Sperm Binding to the Human Zona Is Mediated by a Calcium Influx1

Patricio Morales2,a, Eduardo Pizarroa, Milene Konga, Bredford Kerrb, Francisco Cericb, and Pilar Vigilb

a Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, Antofagasta, Chile b Faculty of Biological Sciences, P. Catholic University of Chile, Santiago, Chile

ABSTRACT

The mechanism by which GnRH increases sperm-zona pellucida binding in humans was investigated in this study. We tested whether GnRH increases sperm-zona binding in Ca2+-free medium and in the presence of Ca2+ channel antagonists. We also examined the GnRH effect on the intracellular free Ca2+ concentration ([Ca2+]i). Sperm treatment with GnRH increased sperm-zona binding 300% but only when Ca2+ was present in the medium. In Ca2+-free medium or in the presence of 400 nM nifedipine, 80 µM diltiazem, or 50 µM verapamil, GnRH did not influence sperm-zona binding. GnRH increased the [Ca2+]i in the sperm in a dose-dependent manner. The maximum effect was reached with 75 nM GnRH. The GnRH-induced increase in [Ca2+]i was fast and transient, from a basal [Ca2+]i of 413 ± 22 nM to a peak value of 797 ± 24 nM. The GnRH-induced increase in [Ca2+]i was entirely due to a Ca2+ influx from the extracellular medium because the increase in [Ca2+]i was blocked by the Ca2+ chelator EGTA and by the Ca2+ channel antagonists nifedipine and diltiazem. These antagonists, however, were not able to inhibit the progesterone-activated Ca2+ influx. On the contrary, T-type calcium channel antagonists pimozide and mibefradil did not affect GnRH-activated Ca2+ influx but inhibited the progesterone-activated Ca2+ influx. Finally, the GnRH-induced Ca2+ influx was blocked by two specific GnRH antagonists, Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH and Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3,6-GnRH. These results suggest that GnRH increases sperm-zona binding via an elevation of [Ca2+]i through T-type, voltage-operated calcium channels.

Fallopian tubes, fertilization, GnRH, sperm capacitation/acrosome reaction

INTRODUCTION

The binding between the sperm and the egg outermost coat, the zona pellucida (ZP), is a crucial event in mammalian fertilization [1]. In most of the mammalian species studied until now, upon binding to the ZP surface, the fertilizing spermatozoon undergoes acrosomal exocytosis [1]. The acrosome reaction, induced by a specific component of the ZP, is necessary for the success of fertilization because it helps the fertilizing spermatozoon to traverse the perivitelline space and to fuse with the oocyte plasma membrane [1]. Therefore, molecules that influence the sperm-ZP binding process have the potential to alter the fertilization outcome.

There is a great amount of work that indicates that progesterone (P) stimulates human sperm acrosome reactions [2–4]. The P-induced acrosomal exocytosis is accompanied by a transient elevation of intracellular free Ca2+ concentration ([Ca2+]i), which is a consequence of a Ca2+ influx [4–6]. Whether there are voltage-operated calcium channels (VOCC) involved in the P-induced Ca2+ entry is still in contention (for recent reviews, see [7–9]). Evidence has been presented suggesting that the P-activated Ca2+ influx and/or the acrosome reaction are sensitive to inhibition of VOCC [3, 10–14]. Other work, however, has failed to demonstrate the involvement of VOCC in these processes [15–19]. If we analyze the evidence that favors an involvement of VOCC in the effect of P on human sperm, the type of channel involved is also in dispute. Two possible types of calcium channels in dispute are the L-type (long lasting, large current) and the T-type (low-voltage activated, transient) [7, 8, 11, 20–24]. The evidence that favors the existence of T-type VOCC in sperm derives mainly from work conducted with mature mice sperm and purified murine spermatogenic cells [8, 20, 22–25]. In experiments conducted with mature human spermatozoa, however, the evidence benefits the involvement of L-type calcium channels [11, 13, 26, 27].

GnRH is a decapeptide of hypothalamic origin that causes the release of LH and FSH upon binding to a specific receptor located on pituitary gonadotrophs. The effect of GnRH is mediated by an increase in the [Ca2+]i via Ca2+ influx [28]. In addition, GnRH or GnRH-like molecules have been found in human follicular fluid [29] and seminal plasma [30, 31]. Local production of GnRH-like molecules has also been reported in the ovary [32] and testis [33, 34], where granulosa and Sertoli cells, respectively, produce it. These findings suggest that GnRH-like molecules may play either or both an endocrine or paracrine function in these extrapituitary tissues [35]. In accord with this, it was shown that GnRH and GnRH agonists enhanced bovine in vitro fertilization through an effect on the cumulus-oocyte complex [36] and that GnRH increased sperm binding to the human ZP by a direct effect on the sperm cells [37]. Moreover, we have shown that GnRH antagonists inhibited sperm-ZP binding in humans in a dose dependent manner [38]. All these observations allowed us to suggest that human spermatozoa might interact with GnRH (or with GnRH-like molecules) during their journey through the male and female genital tracts and that this interaction with GnRH might confer the spermatozoa increased zona-binding capabilities.

The aim of the present work was to characterize the mechanism by which GnRH increases sperm-zona binding in humans. We studied whether the GnRH-induced sperm-zona binding depended upon the presence of extracellular Ca2+ and whether GnRH has any effect on sperm [Ca2+]i. In addition, the effect of calcium channel antagonists on the GnRH-induced sperm-zona binding and [Ca2+]i was evaluated. The involvement of calcium channels in the sperm-zona binding process is discussed.

MATERIALS AND METHODS

Reagents

The following chemicals and reagents were purchased: progesterone, GnRH, nifedipine, diltiazem, verapamil, pimozide, fura 2-AM, and the GnRH antagonist Ac-D-Nal1-Cl-D-Phe2-3-Pyr-D-Ala3-Arg5-D-Glu(AA)6-GnRH (Nal-Glu) from Sigma Chemical Co. (St. Louis, MO); the GnRH antagonist Ac-3,4-dehydro-Pro1,-p-fluoro-D-Phe2, D-Trp3,6-GnRH (4pF) was obtained from Bachem Bioscience Inc. (Torrance, CA); mibefradil was obtained from Hoffman-La Roche (Basel, Switzerland). Deionized water used in these experiments was purified to >=18 M{Omega}-cm with an EASY-pure UV/UF ion-exchange system (Barnstead/Thermolyne, Dubuque, IA).

Source and Preparation of Biological Material

Human zonae pellucidae Human oocytes were dissected from ovarian tissue and stored at -80°C as described elsewhere [39, 40]. Briefly, ovarian tissue was placed on ice and dissected immediately, following the protocol of Overstreet et al. [41]. Zona-intact, immature oocytes were denuded of granulosa cells, placed in capillary tubes containing 2 M dimethylsulfoxide (DMSO) in PBS, and stored at -80°C. After thawing, the oocytes were freed of remaining cumulus cells by passage through a narrow-bore pipet. As a result of freezing and thawing, these oocytes were never viable. Before use, the oocytes were cut to obtain two equal halves, or hemizonae [37, 38, 40, 42, 43].

Sperm suspension preparation Semen samples were obtained from normal donors after 2 to 3 days of sexual abstinence, with the approval of the ethics committee of the University of Antofagasta. All samples had normal semen parameters according to World Health Organization guidelines [44]. The specimens were allowed to liquefy for 30 to 60 min at 37°C in a slide warmer. Motile spermatozoa were selected by centrifugation through a two-step Percoll gradient as described elsewhere [40, 45]. Briefly, aliquots of semen were layered over the upper step of the Percoll gradient and centrifuged for 20 min at 300 x g. The pellet was diluted in 10 ml of modified Tyrode medium consisting of 117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KCl, 25 mM NaHCO3, 2.5 mM CaCl2, 0.5 mM MgCl2, 2 mM glucose, 0.25 mM Na pyruvate, 19 mM Na lactate, 70 µg/ml of both streptomycin and penicillin, phenol red, and 0.3% of BSA [45], centrifuged again at 300 x g for 10 min, and then resuspended in the same medium but with 2.6% BSA. The sperm concentration was estimated using a hemocytometer and then adjusted to 20 x 106 cells/ml. The sperm were then incubated at 37°C in 5% CO2 for 4.5 h to promote capacitation [13, 15, 18, 46]. In some experiments, capacitated sperm were resuspended in nominally Ca2+-free medium, as described elsewhere [39]. This medium consisted of 117.5 mM NaCl, 0.3 mM NaH2PO4, 8.6 mM KCl, 25 mM NaHCO3, 0.5 mM MgCl2, 2 mM glucose, 0.25 mM Na pyruvate, 19 mM Na lactate, 187 µg/ml penicillin, 125 µg/ml streptomycin, and 2.6% BSA, with 2.5 mM SrCl2 to support sperm motility [39]. To dilute the sperm in Ca2+-free medium, an equal volume of Ca2+-free medium without BSA was added, then layered on top of 10 ml of Ca2+-free medium with 2.6% BSA, and centrifuged at 500 x g for 6 min. The supernatant was discarded, and the pellet was resuspended in Ca2+-free medium, as described [39]. Then, the sperm were evaluated in the hemizona assay (see below).

Sperm-ZP binding assay The sperm capacity to bind to the ZP was evaluated by using the hemizona assay [42, 43]. Briefly, 99 µl droplets of spermatozoa, suspended in modified Tyrode medium containing 2.6% BSA were treated by adding 1 µl of test or control solutions under oil in a plastic Petri dish. Then, one hemizona was added to the control sperm droplet, and the matching hemizona was added to the test sperm droplet. Control and test sperm droplets containing hemizonae were incubated for 10 min at 37°C, 5% CO2. After incubation, each hemizona was removed and gently washed with a wide-bore pipette. The tightly bound spermatozoa on the outer surface of each hemizona were counted under a phase-contrast microscope. These procedures have been described in detail elsewhere [37, 38, 40].

Effect of Calcium Channel Antagonists upon Sperm-ZP Binding

To test whether the stimulatory effect of GnRH on sperm-ZP binding required extracellular calcium, the hemizona assay was conducted in nominally Ca2+-free medium and in the presence of several calcium channel antagonists. In one set of experiments, aliquots of capacitated sperm suspensions were incubated with 75 nM GnRH [37] or saline in the presence or absence of 2.5 mM CaCl2 for 5 min and then tested in the hemizona assay. To test the effect of calcium channel antagonists upon GnRH-induced sperm-zona binding, aliquots of capacitated sperm suspensions were treated with nifedipine (400 nM), diltiazem (80 µM), or verapamil (50 µM) alone or followed 5 min later by the addition of 75 nM GnRH. The doses of calcium channel antagonists used in this study are equivalent to those used by others and do not have any deleterious effect on sperm motility [11–13, 15, 18, 19, 47]. After an additional 5 min, the hemizona assay was carried out.

Measurement of [Ca2+]i

Capacitated sperm suspensions were prepared for [Ca2+]i determination by loading with the acetoxy-methyl ester of fura 2 (3 µM final extracellular concentration) for 30 min at 37°C and 5% CO2, essentially as described elsewhere [2, 3, 6, 10]. To remove the free fura 2, the cells were diluted with 10 ml of Tyrode medium 2.6% BSA without phenol red and centrifuged twice at 300 x g for 10 min. Then, the cells were resuspended in Tyrode medium without phenol red at a final concentration of 7–8 x 106 cells/ml and incubated for 30 min at 37°C in 5% CO2. Then, 1-ml sperm aliquots were used for spectrofluorometry resuspending directly into stirred fluorescence cuvettes. All these procedures were carried out in the dark to prevent sample photobleaching.

Fluorescence caused by [Ca2+]i under various experimental conditions was monitored with a Shimadzu model 1501 spectrofluorometer at an excitation wavelength pair of 340/380 nm and emission wavelength of 510 nm. Spectrofluorometry was performed in a methylacrylate cuvette magnetically stirred and warmed to 37°C in a heated cuvette holder. After equilibration for 2 min, measurements of [Ca2+]i were started. At approximately 100 sec after the beginning of each sample run, GnRH (25–100 nM) was added to the sperm suspension. In some experiments, 2.5 mM EGTA (pH 8.0) or 0.25 mM La3+ was added before adding GnRH. To test whether calcium channels were involved in the sperm response to GnRH, sperm aliquots were incubated with the following calcium channel antagonists: diltiazem (2 nM–80 µM), nifedipine (100–400 nM), pimozide (16 µM), or mibefradil (22 µM) for 5 or 15 min at 37°C, 5% CO2, before the addition of 75 nM GnRH. In other experiments, we tested the effect of GnRH antagonists on the sperm response to GnRH. Sperm aliquots were treated with 75 nM Nal-Glu or 4pF for 5 min at 37°C in 5% CO2, before the addition of GnRH (75 nM). Before the end of each experiment, P (0.69 µM) was added as a positive control. Sequential additions of 20 µM digitonin and 10 mM Tris-EGTA were made near the end of each experiment to facilitate determination of [Ca2+]i, as described elsewhere [2, 3, 6, 10], using a dissociation constant of 285 nM for fura 2 at 37°C [10]. The data were corrected for autofluorescence as described [5]. In the experiments described here, GnRH, diltiazem, and verapamil were dissolved in saline; pimozide and P were dissolved in DMSO; nifedipine was dissolved in ethanol; and mibefradil was dissolved in water. The final concentration of ethanol or DMSO in the sperm suspensions never exceeded 0.1%.

Statistical Analysis

The data were analyzed by ANOVA for repeated measures using the StatView program (SAS Institute, Cary, NC) on an Apple Power Macintosh 6500/225. Differences between individual groups were examined with Fisher's protected least significant difference test. Paired comparisons were conducted by a paired t-test, and all data are presented as mean values ± SEM.

RESULTS

As we previously reported, a 5-min preincubation of the cells with 75 nM GnRH increased the number of zona-bound sperm almost threefold (88 ± 8 control vs. 249 ± 15 GnRH; mean ± SEM, P < 0.01; Fig. 1). This effect of GnRH depended upon the presence of Ca2+ in the culture medium. In the absence of extracellular Ca2+, GnRH did not stimulate sperm-zona binding (110 ± 12 control vs. 111 ± 9 GnRH; Fig. 1). Moreover, sperm preincubated with the calcium channel antagonists nifedipine (400 nM), diltiazem (80 µM), or verapamil (50 µM) before treatment with GnRH did not exhibit higher numbers of zona-bound sperm than did the control, solvent-treated sperm (Fig. 2). In addition, sperm treated with calcium channel antagonists before GnRH bound to the ZP significantly less than the GnRH-alone group (Fig. 2; control, 73 ± 3; GnRH, 173 ± 6; nifedipine, 73 ± 4; nifedipine + GnRH, 89 ± 4; diltiazem, 62 ± 4; diltiazem + GnRH, 65 ± 4; verapamil, 78 ± 4; verapamil + GnRH, 77 ± 4).



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  		FIG. 1. Effect of GnRH on sperm-zona pellucida binding in the presence or absence of extracellular calcium. Sperm capacitated for 4.5 h were treated for 5 min with saline (control) or with 75 nM GnRH in the presence (+Ca2+) or absence (-Ca2+) of 2.5 mM CaCl2 before the hemizona assay. The results are expressed as a percentage (mean ± SEM) of the control hemizonae, and they represent the average ± SEM of six experiments conducted with five different semen samples



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  		FIG. 2. Effect of calcium channel antagonists on the GnRH-induced sperm-zona binding. Sperm capacitated for 4.5 h were treated with solvent (control), 400 nM nifedipine, 80 µM diltiazem, or 50 µM verapamil alone or followed 5 min later by the addition of 75 nM GnRH. The results are expressed as a percentage (mean ± SEM) of the control hemizona. The number of experiments is indicated within each bar. * Significantly higher from all other groups, P < 0.001

Regarding the effect on [Ca2+]i, the results show that GnRH increased the free cytosolic calcium in the sperm cells. This effect depended upon the concentration of GnRH used. Thus, there were differences in the peak and plateau values obtained with different doses of GnRH (Fig. 3). The maximum effect was reached with 75 nM GnRH. Lower and higher doses were less effective (Fig. 3, A and B). Similar results were obtained regarding the ability of GnRH to increase sperm-zona binding [37]. The GnRH-induced increase in [Ca2+]i was fast and transient, and with 75 nM, this increase was from a basal value of 413 ± 22 nM to a peak value of 797 ± 24 nM (Fig. 3B). This represents a stimulation of 194 ± 8% in comparison to the basal calcium level (Fig. 3B, insert). The peak value was reached about 20 sec after GnRH addition and slowly came back to a new basal value, slightly higher than before, about 100 sec later (Fig. 3A). The baseline Ca2+ levels in this study, slightly higher than that reported by others [6, 10], may be related to the use of sperm capacitated for 4.5 h instead of 24 h [6, 10]. In all experiments performed, 0.69 µM P was added at the end of each run as a positive control. As has been described by others [2–4, 6, 10], P provoked a rapid and transient increase in the [Ca2+]i (data not shown). This concentration of P provoked an increase in [Ca2+]i that was always of greater magnitude than that induced by 75 nM GnRH (194 ± 8% for GnRH vs. 295 ± 13% for P, P < 0.01).



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  		FIG. 3. Effect of GnRH on the [Ca2+]i in capacitated human sperm measured using fura 2. In A, the effect of 25, 50, 75, and 100 nM GnRH on the [Ca2+]i is shown. GnRH was dissolved in saline and added (arrow) at 100 sec. A representative experiment of five is shown. In B, the mean ± SEM (n = 5) of the basal (filled bars) and peak (empty bars) calcium levels for 25, 50, 75, and 100 nM GnRH is shown. In the insert, the percentage increase over the basal value for each GnRH concentration is shown

The effect of GnRH upon [Ca2+]i on spermatozoa was inhibited by the prior addition of 2.5 mM EGTA or 0.25 mM La3+ (Fig. 4). When Ca2+ (2.5 mM) was added back to the EGTA-treated sperm suspension, there was an immediate rise in [Ca2+]i (Fig. 4). As described by others, EGTA and La3+ also blocked the effect of P (data not shown).



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  		FIG. 4. Effect of EGTA and La3+ on the ability of GnRH (75 nM) to increase [Ca2+]i in capacitated human sperm measured using fura 2. Fura 2-loaded sperm were treated with 75 nM GnRH at 100 sec (arrow) in the presence of 2.5 mM Ca2+, 2.5 mM EGTA (added 1 min earlier), or 0.25 mM La3+ (added 1 min earlier). To the EGTA-treated cells at 200 sec, 2.5 mM Ca2+ was added back. A representative experiment of five is shown

The GnRH-induced increase in [Ca2+]i was also blocked by the calcium channel antagonists nifedipine and diltiazem (Fig. 5). Four hundred nM nifedipine inhibited the effect of 75 nM GnRH by 89 ± 6% after a 5-min preincubation and by 100% after a 15-min preincubation (Table 1). A lower dose of nifedipine (100 nM), however, was unable to block the GnRH-induced Ca2+ influx, even after a 15-min preincubation period (Table 1). Diltiazem also inhibited the GnRH-induced increase in [Ca2+]i (Fig. 5). The inhibition was more effective when the sperm were preincubated for 15 min than for 5 min (88 ± 5% vs. 33 ± 6%; Table 1). However, the inhibition with diltiazem never reached 100%, even with a dose as high as 80 µM (Table 1). On the other hand, neither nifedipine nor diltiazem, used at the same concentrations that were effective to block the GnRH effect, were able to inhibit the P-induced increase in [Ca2+]i (Fig. 5).



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  		FIG. 5. Effect of the calcium channel antagonists nifedipine and diltiazem on the ability of GnRH and progesterone to increase [Ca2+]i in capacitated human sperm measured using fura 2. Fura 2-loaded sperm were treated with 75 nM GnRH at 100 sec (arrow) in the presence of 400 nM nifedipine or 80 µM diltiazem (both added 15 min earlier). In all the experiments, 0.69 µM progesterone (P) was added at 200 sec. A representative experiment of five is shown


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  		TABLE 1. Inhibitory effect of nifedipine and diltiazem on the GnRH-induced increase in [Ca2+]i

We also tested the effect of two calcium channel antagonists, pimozide and mibefradil, that have been described as having primarily T-channel selectivity. Neither pimozide nor mibefradil, used at µM concentration, were effective in inhibiting the GnRH-induced [Ca2+]i (Fig. 6A); however, both calcium channel antagonists inhibited the P-induced [Ca2+]i by 62 ± 6% and 68 ± 9%, respectively (Fig. 6B).



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  		FIG. 6. Effect of the calcium channel antagonists pimozide and mibefradil on the ability of GnRH (A) and progesterone (B) to increase [Ca2+]i in capacitated human sperm measured using fura 2. Fura 2-loaded sperm were treated with 75 nM GnRH in the presence of 16 µM pimozide or 22 µM mibefradil (both added 5 min earlier). At 200 sec, 0.69 µM progesterone (P) was added. The results are expressed as a percentage of the control and they represent the mean ± SEM (n = 6). * Significantly higher than the other groups, P < 0.001

Finally, the GnRH-induced increase in [Ca2+]i was blocked by a 5-min preincubation with 75 nM of the GnRH antagonists 4pF and Nal-Glu (Fig. 7). These GnRH antagonists inhibited the increase in intracellular calcium by 86 ± 9% and 80 ± 7% (Nal-Glu and 4pF, respectively). The GnRH antagonists, however, did not inhibit the increase in [Ca2+]i provoked by P (Fig. 7).



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  		FIG. 7. Effect of GnRH antagonists on the ability of GnRH (75 nM) to increase [Ca2+]i in capacitated human sperm measured using fura 2. Fura 2-loaded sperm were treated with 75 nM GnRH at 100 sec (arrow) in the presence of 75 nM Nal-Glu or 4-pF (both added 5 min earlier). In all the experiments, 0.69 µM progesterone (P) was added at 200 sec. A representative experiment of five is shown

DISCUSSION

In this work, we have extended our previous observations concerning the effect of GnRH on human sperm-ZP binding [37, 38]. Here we show that the GnRH-induced increase in sperm-zona binding depends upon the presence of extracellular Ca2+ and that calcium channel antagonists inhibit this effect of GnRH. In addition, we have found that GnRH causes an increase in the [Ca2+]i in human sperm. The results with EGTA and La3+ as well as those with calcium channel antagonists suggest that this increase in [Ca2+]i is due to a Ca2+ influx through VOCC. To our knowledge, this is the first time that it has been shown that GnRH can cause an increase in the [Ca2+]i in spermatozoa.

Our results are in accord with the GnRH-induced increase in [Ca2+]i in pituitary gonadotrophs [28, 48–51]. In pituitary gonadotrophs, the activation of GnRH receptors leads to rapid increases in inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, with the consequent onset of Ca2+ signaling and translocation of protein kinase C to the plasma membrane [49–51]. The cytoplasmic Ca2+ response to GnRH in pituitary cells is biphasic, with an immediate spike phase and a subsequent plateau phase. The initial spike phase is derived primarily by Ca2+ mobilization from intracellular stores by IP3. However, the capacity of the IP3-sensitive calcium pools is limited, and sustained agonist stimulation requires continuous calcium entry across the plasma membrane to maintain the less prominent but functionally important plateau phase of the calcium response. The LH response to GnRH also occurs in a biphasic manner that coincides with the GnRH-induced calcium signal, and its prolonged plateau phase is likewise dependent on the presence of extracellular calcium [48–50].

In pituitary gonadotrophs, GnRH concentrations equivalent to those used in our study provoked a similar response to that seen by us in human spermatozoa, both in terms of the peak value and the amplitude of the curve [52–54]. The main difference between our results and those studies with gonadotrophs is that in the gonadotrophs, the initial increase in [Ca2+]i is independent of extracellular Ca2+ [52–54]. In pituitary gonadotrophs, the initial phase of the increase in [Ca2+]i in response to GnRH is independent of extracellular Ca2+, whereas the sustained phase is dependent on Ca2+ entry through voltage-sensitive and -insensitive Ca2+ channels [48, 55, 56]. The extracellular Ca2+ independence of the initial Ca2+ response reflects the release of Ca2+ from intracellular sources, presumably by IP3-mediated Ca2+ release from the endoplasmic reticulum [28]. However, when the extracellular calcium is chelated with EGTA or Ca2+ channels are blocked with D600 (methoxyverapamil), GnRH-stimulated LH exocytosis is also blocked [57]. This situation is similar to the mammalian sperm acrosome reaction, in which exocytosis of the acrosome does not take place without external Ca2+ [58]. Thus, in gonadotrophs, although the initial raise in the [Ca2+]i provoked by GnRH still occurs in the absence of extracellular Ca2+ or during blockade of Ca2+ influx, LH release does not take place. Other than pituitary gonadotrophs, it has been shown that GnRH can increase the [Ca2+]i in Leydig cells [59], ovarian granulosa cells [60], and placental cells [61].

In mammalian sperm, there are results that indicate that phosphoinositide hydrolysis to produce inositol phosphates is completely dependent upon the presence of extracellular Ca2+ [19, 62]. The same seems to be true for the generation of diacylglycerol [63]. These results tend to neglect a role for phosphoinositide metabolism in the control of Ca2+ influx in mammalian spermatozoa. Recent reports, however, strongly suggest that there may be a role for the mobilization of intracellular Ca2+ in sperm capacitation and the acrosome reaction. It was shown that thapsigargin, a highly specific inhibitor of the Ca2+-ATPase of intracellular, nonmitochondrial membranes [64], causes fast elevation of [Ca2+]i and acrosome reactions in mammalian sperm [65–68]. In addition, IP3 receptors have been localized in the sperm acrosome of several mammalian species [65, 68, 69]. In somatic cells, ligand-mediated generation of IP3 followed by calcium release from IP3-gated internal stores promotes extracellular Ca2+ influx across the plasma membrane [70]. This phenomenon of capacitative calcium entry is not completely understood, but calcium depletion from the internal stores seems to be the signal for the opening of calcium channels [71]. Additional work is needed to resolve whether mammalian sperm behave differently from somatic cells in which the generation of IP3 and diacylglycerol precedes the increase in [Ca2+]i.

In addition, our results are in accord with the P-induced Ca2+ influx in human sperm [2, 3, 6, 10, 15, 18]. The responses of the sperm to GnRH and P were comparable in terms of the shape of the curve; however, 0.69 µM P provoked an increase in [Ca2+]i that was of greater magnitude than that provoked by all the doses of GnRH tested. Preliminary results indicated that when sperm were exposed first to P and then to GnRH, the increase in [Ca2+]i was greater than that seen when the hormones were presented in the inverse order (unpublished results). This is in agreement with the acrosomal exocytosis of mouse sperm in response to the sequential addition of P and ZP [63] and the regulation of GnRH action by gonadal steroids in gonadotrophs [72–74].

Regarding the involvement of VOCC on the GnRH-induced calcium influx, our results suggest the participation of dihydropyridine-sensitive channels in this process. Although the existence of L-type calcium channels in mammalian sperm has not yet been confirmed, work from Benoff and colleagues suggests that mammalian sperm possess a unique isoform of an L-type, dihydropyridine-sensitive VOCC [7, 11, 21, 27]. In our work, calcium influx in response to GnRH was blocked by nanomolar concentration of nifedipine and diltiazem. In contrast, inhibition of the GnRH-induced increase in [Ca2+]i was not observed using the calcium channel antagonists with T-channel selectivity, pimozide and mibefradil. Neither of the two agents was effective in inhibiting the response of the sperm cells to GnRH. Thus, the evidence here presented suggests that the mechanism of action of the GnRH-induced Ca2+ influx pathway includes the activation of L-type calcium channels.

Regarding the participation of VOCC on the P-induced calcium influx and/or the acrosome reaction in human sperm, there are contradictory findings. There are several reports that indicate that these events are not susceptible to inhibition by verapamil, diltiazem, and nifedipine [14, 15, 18, 19] or are only slightly sensitive (less than 30% inhibition) [3]. Other reports showed that a prolonged exposition of the sperm (48 h) or a very large concentration of the antagonists (200 µM), exceeding the concentration required to block VOCC, were necessary for them to be effective [11, 12]. On the other hand, several studies indicated that VOCC are involved in the P-induced Ca2+ influx, the acrosome reaction, or both in human sperm. In one of them, the P-stimulated acrosome reaction was inhibited by 10–100 nM nifedipine [13]. In a recent study, Garcia and Meizel showed that the P-stimulated Ca2+ influx was inhibited by two inhibitors of T-type VOCC, mibefradil and pimozide [10]. A similar observation was reported by Shiomi et al., using human follicular fluid as the Ca2+-influx stimulus [14]. In the Garcia and Meizel [10] study, however, the results regarding the acrosome reaction were contradictory. Although one Ca2+ channel antagonist increased the percentage of acrosome-reacted sperm, the other significantly inhibited it. On the other hand, Blackmore and Blackmore and Eisoldt failed to find inhibition of the P-stimulated Ca2+ influx by mibefradil [16, 17]. These contradictory results highlight the difficulty of characterizing the P-activated Ca2+ influx pathway in human sperm.

In our study, the P-induced increase in [Ca2+]i was only susceptible to inhibition by T-type calcium channel antagonists; this was the case in a way that was very similar to that described by Garcia and Meizel [10]. Garcia and Meizel found that 16 µM pimozide and 22 µM mibefradil inhibited the P-induced calcium influx by 75% and 65%, respectively. In our study, these figures were 62% and 68%, respectively. Moreover, Shiomi et al. [14] showed that calcium influx stimulated by human follicular fluid was inhibited by the T-type calcium channel blocker amiloride. Unlike others, however, we did not find inhibition of the P-activated calcium influx by the VOCC antagonists nifedipine and verapamil [3, 11–13]. Thus, our work agrees with those reports describing that neither verapamil [14, 15, 18, 19] nor diltiazem or nifedipine [15] inhibited the P-induced increase in [Ca2+]i.

The differences found in the participation of L-type and T-type VOCC in the increase in [Ca2+]i induced by GnRH and progesterone, respectively, may reflect differences in their mechanism of action. GnRH causes an increase in sperm-zona binding and does not affect acrosome reactions [37]. On the other hand, progesterone has a clear acrosome reaction-inducing effect [2–4].

In conclusion, this study has demonstrated that the ability of GnRH to increase sperm-zona binding depends upon Ca2+ influx and that the most probable mechanism of Ca2+ entry through the sperm plasma membrane is via VOCC of the L-type. How this GnRH-induced increase in [Ca2+]i is related to a higher ability of the sperm to bind to the ZP is still a matter of study.

ACKNOWLEDGMENTS

We thank Miguel Llanos for his valuable comments.

FOOTNOTES

First decision: 3 March 2000.

1 This work was financed by Fondecyt 197 1243. This investigation received financial support from the UNDP/UNFPA/WHO/World Bank Special Programme of Research, Development, and Research Training in Human Reproduction, World Health Organization, as project 98184. Back

2 Correspondence: Patricio Morales, Unit of Reproductive Biology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile. FAX: 5655 637 802; pmorales{at}uantof.cl Back

Accepted: March 30, 2000.

Received: January 31, 2000.

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