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Biology of Reproduction 63, 57-63 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Steroidal Sigma Receptor Ligands Affect Signaling Pathways in Human Spermatozoa1

Michael Schaefera, Ursula F. Habenichtb, Mathias Bräutigamb, and Thomas Gudermann2,,3,a

a Institut für Pharmakologie, Freie Universität Berlin, D-14195 Berlin, Germany b Forschungslaboratorien der Schering AG, D-13342 Berlin, Germany

ABSTRACT

In human spermatozoa, Ca2+ entry is stimulated by progesterone or prostaglandin E1 (PGE1). The regulation of cation currents by progestins involves sigma receptors, and sigma binding sites are abundant in testis. We examined the effects of sigma ligands on human spermatozoa. Ca2+ entry induced by progesterone or PGE1 was not altered by the sigma ligands haloperidol and ditolylguanidine. However, the steroidal sigma ligands RU 3117 and RU 1968 had distinct effects. Stimulation by RU 3117 resulted in activation and homologous desensitization of the sperm progesterone receptor but not of the PGE1 receptor. Because haloperidol and ditolylguanidine did not affect RU 3117 and progesterone actions in spermatozoa, we conclude that sigma receptors are not involved. However, RU 1968 potently inhibited both the progesterone- and PGE1-induced Ca2+ entry and acrosome reaction. At higher concentrations, RU 1968 also inhibited hormonal Ca2+ signaling in fibroblasts. Despite suppression of Ca2+ mobilization, inhibition of phospholipase C by RU 1968 was not observed. Furthermore, RU 1968 did not impair the binding of inositol-1,4,5-trisphosphate to its endoplasmic reticulum receptor. Because RU 1968 preferentially inhibits signaling pathways in spermatozoa, the future development of more selective drugs structurally related to RU 1968 may be a novel approach for pharmacological contraception.

calcium, signal transducers, sperm, sperm capacitation/acrosome reaction, testes

INTRODUCTION

Apart from ZP3, a protein component of the zona pellucida, E-type prostaglandins and progesterone mediate calcium entry and acrosome reaction in human spermatozoa [1–4]. Little is known about the molecular mechanism underlying progesterone-induced Ca2+ influx in human spermatozoa [5]. The target protein by which progesterone stimulates Ca2+ entry is an ill-defined binding site located in the sperm head region [6, 7]. Progestins are known to regulate ion channels by interaction with various membrane receptors [8]. Currently known examples for such nongenomic steroid actions are the allosteric potentiation of GABAA receptor/Cl- channel activity by progesterone [9, 10] and the modulation of ion channels via binding of several neurosteroids to sigma receptors [11, 12]. The pharmacological profile of the extragenomic sperm progesterone receptor is not compatible with that of the neuronal GABAA receptor/Cl- channels [13]. Therefore, we wondered whether the sperm progesterone receptor may be related to sigma receptors.

Progesterone binds to sigma receptors and may represent a physiological endogenous ligand [14, 15]. Based on pharmacological differences, two types of sigma receptors, {sigma}1 and {sigma}2, are currently defined [16]. Both sigma receptor subtypes bind progesterone with an affinity in the upper nanomolar range [15, 17], which is well within the concentration range of progesterone acting on human spermatozoa. The purified and cloned {sigma}1 receptor binds progesterone with an affinity similar to that of native sigma binding sites in liver microsomal membranes [17]. The marked structural and pharmacological similarity of the cloned {sigma}1 receptor with yeast sterol isomerases may point to a functional role of steroid binding to the {sigma}1 receptor [17, 18]. The {sigma}2 receptor has not yet been identified at the molecular level. A regulation of ligand-operated ion channels by steroidal sigma receptor ligands such as progesterone and other neurosteroids has been described for several cellular systems. The modulation of N-methyl-D-aspartate (NMDA)-evoked responses in hippocampal neurons by the neurosteroid dehydroepiandrosterone (DHEA) supposedly involves {sigma}1 receptors [19]. Consistent with this report, Monnet et al. [11] demonstrated that the NMDA-evoked release of norepinephrine from hippocampal slices is potentiated by DHEA and inhibited by pregnenolone sulfate via sigma receptors. In adrenal chromaffin cells, the nicotine-stimulated catecholamine release is attenuated by {sigma}1 receptor ligands [20]. Other functional effects of sigma receptor ligands (for review, see [15, 21]) include the modulation of ion channels in NCB-20 cells via binding to a low-affinity sigma receptor [22] and the inhibition of phosphoinositide turnover in brain synaptoneurosomes [23]. Recently, G protein-dependent [24] and -independent [25] inhibition of voltage-activated potassium channels via {sigma}1 receptors was observed. In the rat brain stem, activation of the cognate {sigma}1 receptor [17] results in its translocation from the cytosol to the cell membrane where it contributes to the activation of phospholipase C ß1 and ß2 isoforms, resulting in a decrease in hypoglossal activity [26].

Sigma receptors have been detected in a variety of peripheral tissues, including testis, epididymis, liver, adrenal gland, placenta, and ovary [27]. In male gonads, the localization of sigma receptors is restricted to the seminiferous tubules and to the tubular system of the epididymis [28]. Binding sites were not detected in the interstitial area of the testis. The high potency of (+)-SKF 10 047 and of (+)-pentazocine to displace [3H]haloperidol from guinea pig testis membranes [27] and the reduction of binding sites after prolonged exposure to haloperidol in rat testes [29] indicate that a major portion of the {sigma} receptors in testis belong to the {sigma}1 subtype. The binding sites for steroidal sigma ligands in rat testis membranes were further characterized by Bonfils et al. [30]. A (+)-PPP binding site in rat testis membranes was competed for by haloperidol, 1,3-di-(2-tolyl)guanidine (DTG), progesterone, and the steroidal sigma ligands RU 1968 and RU 3117. In rat testis membranes, steroidal sigma ligands (RU 1968 and RU 3117) interacted with multiple sites, and binding was partially displaced by haloperidol, benztropine, and progesterone [30]. However, the functional consequences of RU 1968 or RU 3117 binding to rat testis membranes remain obscure.

In the present study, we wanted to determine whether sigma receptors would be involved in the stimulation of Ca2+ influx by progesterone and PGE1 in human spermatozoa. Classical high-affinity sigma ligands such as haloperidol and DTG did not affect [Ca2+]i homeostasis in resting or stimulated human spermatozoa. The steroidal sigma ligands RU 3117 and RU 1968 had opposite effects on human spermatozoa: Whereas RU 3117 induced a rapid Ca2+ influx by stimulating the extragenomic progesterone receptor, RU 1968 was a potent inhibitor of human sperm activation. RU 1968 potently blocked progesterone- and PGE1-elicited Ca2+ entry and hormone-induced acrosomal exocytosis.

MATERIALS AND METHODS

Chemicals

Fluo 3AM was obtained from Molecular Probes (Eugene, OR). Progesterone, PGE1, haloperidol, progesterone, (+)-PPP, Triton X-100 reduced form, bisbenzimide, and fluorescein isothiocyanate (FITC)-conjugated Pisum sativum agglutinin were obtained from Sigma (Deisenhofen, Germany). DTG was from RBI (Natick, MA). The steroidal sigma ligands RU 3117 and RU 1968 were provided by Schering AG (Berlin, Germany). [3H]Myo-inositol-1,4,5-trisposphate (InsP3, 21 Ci/mmol) was from NEN (Bad Homburg, Germany). All other reagents were of analytical grade. Stock solutions of steroids (10–100 mM) and of PGE1 (10 mM) were prepared in absolute ethanol and freshly diluted prior to each experiment. Final concentrations of ethanol did not exceed 0.1%.

Preparation of Sperm Suspensions and Cell Culture

Ejaculates were obtained from healthy volunteers, and semen samples with normal parameters for sperm count, viability, and motility were pooled. Motile spermatozoa were enriched by a swim-up procedure. Liquefied semen (200–400 µl) was sublayered to a modified BWW medium [3] containing 166 mM NaCl, 5 mM KCl, 1.3 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5.5 mM glucose, 0.25 mM sodium pyruvate, 21 mM lactic acid, 25 mM NaHCO3, and 20 mM Hepes, pH 7.4, supplemented with 10% (v/v) fetal calf serum (BWW-FCS). Swim-up was allowed to proceed for 60–120 min at 37°C in a humidified atmosphere containing 5% CO2. Motile sperm were recovered from the supernatant by centrifugation (600 x g, 8 min) and resuspended in BWW-FCS. To analyze acrosomal exocytosis, an additional incubation step (4–6 h, 37°C, 5% CO2 in a humidified atmosphere) was performed to promote capacitation of spermatozoa. Prior to the experiments, spermatozoa were washed and resuspended in BWW to a final concentration of 2–5 x 106 sperm/ml.

Mouse Ltk- cells stably transfected with the m5 muscarinic receptor (LM5 cells) were maintained as described previously [31]. All experiments using LM5 cells were performed in Hepes-buffered saline (HBS) containing 142 mM NaCl, 6 mM KCl, 1.25 mM MgCl2, 1.25 mM CaCl2, 5.5 mM glucose, 10 mM Hepes, pH 7.4, and 0.2% BSA (w/v).

Measurement of Intracellular Free Ca2+ Concentrations

Sperm suspensions (2–10 x 106 cells/ml) and LM5 cells (2 x 106 cells/ml) were loaded with fluo 3AM at 2.5 µM for 30 min at 37°C and washed in BWW and HBS, respectively. Cell suspensions were placed in a cuvette holder (37°C) of a fluorescence spectrophotometer (LS50B, Perkin Elmer, Norwalk, CT). Fluo 3 was excited at 506 nm, and emission was detected at 526 nm at 250- to 500-msec intervals. Each measurement was followed by a calibration procedure. Fmax was determined after lysis of cells by addition of 0.1% Triton X-100 reduced form; Fmin was obtained by the addition of 20 mM EGTA, pH 8.0. The KD of the fluo 3-Ca2+ complex was assumed to be 400 nM at 37°C as previously described [32]. The intracellular Ca2+ concentrations were calculated by applying the formula [Ca2+]i = KD x (F - Fmin)/(Fmax - F) to the stored data. At the concentrations applied, none of the compounds and solvents displayed any detectable fluorescence that might interfere with the measurement of [Ca2+]i.

Determination of InsP3 Concentrations in LM5 Cells

LM5 cells were seeded in six-well culture dishes and grown to confluency. The growth medium was replaced by 1 ml of prewarmed HBS. After an equilibration period of 15 min, sigma ligands or solvent were added, and cells were incubated for additional 10 min at 37°C. Carbachol (100 µM) or HBS was added up to a final volume of 1.5 ml/well, and inositol phosphates were extracted after 30 sec by exchanging the medium for 1 ml of ice-cold trichloroacetic acid (10%, w/v). The suspension was transferred to microcentrifuge tubes, and cellular debris was removed by centrifugation (12 000 x g, 5 min, at 4°C). Supernatants were extracted with diethyl ether and neutralized by the addition of 30–40 µl of NaHCO3 (1 M). InsP3 concentrations were determined by a radioligand competition assay [33]. Binding of the radioligand was performed for 20 min on ice in a binding buffer containing 100 mM Tris/HCl, pH 9.0, 4 mM EDTA, 4 mg/ml BSA. The binding medium was sublayered by 400 µl of a sucrose solution (5%, w/v, in binding buffer and water, 1:4). Bound radioligand was recovered by centrifugation of membranes through the sucrose layer (12 000 x g, 10 min, 4°C). Radioactivity was determined by liquid scintillation spectrometry.

Detection of Acrosome-Reacted Spermatozoa and Sperm Viability

The percentage of acrosome-reacted spermatozoa was evaluated by a double staining procedure with bisbenzimide and FITC-conjugated Pisum sativum agglutinin as described previously [34]. Capacitated spermatozoa were incubated for 30 min in the presence of agonists or solvent in BWW-FCS as indicated. Staining patterns were assessed with a LSM 510 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) using the 488-nm wavelength of an argon laser for excitation of FITC and the 364-nm wavelength of an ultraviolet laser for excitation of bisbenzimide-DNA. Emitted light was filtered through a 505-nm longpass filter for FITC fluorescence and a 385-nm longpass filter for bisbenzimide-DNA. The pinholes were adjusted to recover emitted light from a 3-nm plane corresponding to the average diameter of human sperm heads. Staining patterns were evaluated for 200 bisbenzimide-negative spermatozoa per data point. Data are expressed as mean ± SEM percentages of live acrosome-reacted spermatozoa in six to nine samples for two independent experiments.

RESULTS

Ca2+ Homeostasis in Human Spermatozoa and in LM5 Cells

The steroidal sigma ligand RU 3117 (10–30 µM) induced a concentration-dependent elevation of [Ca2+]i in sperm, with kinetic properties similar to the [Ca2+]i signals observed after stimulation with progesterone or PGE1 (Fig. 1A). Treatment of spermatozoa with RU 3117 at 30 µM (n = 6) elevated [Ca2+]i from 108 ± 12 nM to 241 ± 27 nM, and progesterone at 1 µM raised the mean [Ca2+]i from 106 ± 5 nM to 701 ± 54 nM (n = 9). The half-maximal effective concentration of RU 3117 was more than 10 µM, and a saturating effect was not observed at concentrations up to 50 µM (Fig. 1, A and B). Both the potency and the efficacy of progesterone to induce increases in [Ca2+]i were higher than those of RU 3117 (Fig. 2).



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  		FIG. 1. Desensitization of the sperm progesterone receptor by RU 3117. A) Time course of intracellular free Ca2+ concentrations ([Ca2+]i) in fluo 3-loaded human sperm suspensions. Spermatozoa were stimulated by addition of progesterone (1 µM) and prostaglandin E1 (PGE1, 1 µM) for the indicated time intervals. The effects of different concentrations of RU 3117 on [Ca2+]i signals induced by subsequent stimulation with progesterone and PGE1 are shown. B) Concentration-dependent desensitization of progesterone responses by RU 3117. [Ca2+]i transients in human sperm suspensions were induced by the subsequent addition of RU 3117 (solid bars) and progesterone (open bars). Data for peak values after addition of the respective agonist are normalized to the respones elicited by 1 µM progesterone alone.



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  		FIG. 2. Concentration-response relationship of RU 3117 compared with progesterone. For the assessment of concentration-reponse relationships, fluo 3-loaded sperm suspensions were stimulated with either progesterone or RU 3117 at the indicated concentrations. Basal values for [Ca2+]i were subtracted, and data were normalized to the responses induced by 10 µM progesterone. Peak values of the transient increases in [Ca2+]i were determined and used to construct concentration-response curves for progesterone and RU 3117

Treatment of cells with RU 3117 prior to the addition of progesterone and PGE1 resulted in a concentration-dependent desensitization of the extragenomic progesterone receptor but not of the sperm PGE receptor (Fig. 1A). Both peak values of [Ca2+]i transients and areas under the curve after the addition of PGE1 (1 µM) remained unaffected in the presence of 10–30 µM RU 3117. The areas under the curves for PGE1 were determined by integrating the [Ca2+]i concentrations over a 60-sec interval starting from the addition of PGE1. The resulting values are 467.7 nM x min for the solvent, 513.8 nM x min for 10 µM RU 3117, and 473.9 nM x min for 30 µM RU 3117. In contrast, RU 3117 failed to induce a rise of [Ca2+]i in human sperm suspensions that were prestimulated with progesterone at 1 µM (Fig. 3). Preincubation of spermatozoa with PGE1 at 1 µM did not reduce the responses elicited by RU 3117 (data not shown). In contrast to the steroidal sigma ligand RU 3117, the high-affinity sigma ligands haloperidol and DTG neither induced rises of [Ca2+]i nor inhibited the RU 3117- or progesterone-induced Ca2+ entry into human spermatozoa (Fig. 4).



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  		FIG. 3. RU 3117 effects after prestimulation of spermatozoa with progesterone. [Ca2+]i was determined in fluo 3-loaded human sperm suspensions. Spermatozoa were stimulated by progesterone (1 µM), followed by the addition of RU 3117 (30 µM) and PGE1 (1 µM)



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  		FIG. 4. Effects of high-affinity sigma receptor ligands on Ca2+ homeostasis in human spermatozoa. The nondiscriminant, high-affinity sigma receptor ligands haloperidol (1 µM) and DTG (10 µM) or solvent were added prior to a sequential stimulation with 10 µM RU 3117 and 1 µM progesterone. Traces of [Ca2+]i are superimposed

In contrast to RU 3117, the steroidal sigma ligand RU 1968 did not evoke significant increases in [Ca2+]i in human spermatozoa when applied at concentrations up to 30 µM (Fig. 5A). However, progesterone- or PGE1-stimulated increases in [Ca2+]i were suppressed in a concentration-dependent manner by preincubation with RU 1968 (Fig. 5A). A half-maximal reduction of the progesterone- and PGE1-induced Ca2+ peaks was caused by 0.5–0.8 µM RU 1968 (Fig. 5B).



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  		FIG. 5. Inhibition of agonist-mediated [Ca2+]i signaling in human spermatozoa and in LM5 cells by RU 1968. A) Human sperm suspensions were incubated with solvent or with the indicated concentrations of RU 1968 and then stimulated by successive addition of progesterone and PGE1. Time courses of [Ca2+]i under the respective conditions are depicted. B) Concentration-dependent inhibition by RU 1968 of progesterone-induced (solid circles) and PGE1-induced (open circles) [Ca2+]i signals in spermatozoa. The corresponding IC50 values of RU 1968 were 800 nM and 650 nM, respectively. In a mouse fibroblast cell line stably transfected with the m5 muscarinic receptor (LM5 cells), increases in [Ca2+]i were stimulated by carbachol (100 µM). The effects of various concentrations of RU 1968 on [Ca2+]i signaling in LM5 cells are shown (solid triangles). The corresponding IC50 value of RU 1968 was 4 µM. Data are expressed as percentages of the agonist-induced [Ca2+]i increases in cell suspensions pretreated with the solvent alone

Figure 6 illustrates the development of the inhibitory effects of submaximal effective concentrations of RU 1968 over time. Inhibitory properties of RU 1968 (2 µM) were discernible within less than 10 sec. By fitting a first order kinetic model to the data, the calculated time constant and the maximal inhibition were around 18 sec and 74%, respectively. An equilibrium was reached after 2–4 min of pretreatment (Fig. 6).



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  		FIG. 6. Time-dependent development of inhibitory properties of RU 1968. Human spermatozoa were preincubated with RU 1968 (2 µM) for different time intervals. The data represent percentages of progesterone-induced (1 µM) increases in [Ca2+]i compared with pretreatment with the solvent. For the measurement of the effects of RU 1968 without preincubation, a mixture of 1 µM progesterone and 2 µM RU 1968 was applied to the sperm suspension

Because the inhibitory properties of RU 1968 were nondiscriminant between progesterone and PGE receptors, we tested whether Ca2+ signaling in LM5 cells expressing the Gq/11-coupling m5 muscarinic receptor was also affected. When applied at high micromolar concentrations, RU 1968 completely inhibited rises in [Ca2+]i induced by carbachol (100 µM; Fig. 5B) and lysophosphatidic acid (10 µM; data not shown). Because the block of carbachol-mediated Ca2+ transients by RU 1968 was also evident in Ca2+-free HBS medium containing the Ca2+ chelator EGTA (1 mM; Fig. 7), we concluded that the Ca2+ release from internal stores is prevented by pretreatment with RU 1968. The effects of RU 1968 on agonist-mediated [Ca2+]i signaling in LM5 cells may have been caused by the inhibition of phospholipase C. Therefore, agonist-induced formation of InsP3 was determined in LM5 cells.



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  		FIG. 7. RU 1968 blocks Ca2+ release from internal stores in a concentration-dependent manner. Fibroblasts stably expressing a muscarinic acetylcholine receptor were loaded with the Ca2+ indicator and washed in a modified medium containing 100 µM CaCl2. The remaining extracellular Ca2+ was removed by the addition of a Ca2+ chelator (1 mM EGTA) before the experiment. Cells were preincubated in the presence of solvent (bold black line) or 3 µM (thin black line), 10 µM (bold gray line), or 30 µM (thin gray line) RU 1968 and then stimulated by the addition of 100 µM carbachol (horizontal bar)

Phosphoinositide Turnover in Mammalian Fibroblasts

In LM5 cells, stimulation of the m5 muscarinic receptor by 100 µM carbachol elicited an increase in InsP3 concentrations from 3.9 ± 3.1 pmol/well to 104 ± 13.1 pmol/well (Fig. 8). Pretreatment with RU 1968 resulted in slightly elevated InsP3 concentrations in resting cells, increasing from 2.1 ± 2.8 pmol/well in the presence of solvent (0.1% ethanol in HBS) to 10.5 ± 3.6 pmol/well in the presence of 100 µM RU 1968. Upon carbachol stimulation, RU 1968 did not significantly affect InsP3 production (123 ± 20.9 pmol/well in the presence of the solvent versus 114 ± 15 pmol/well in the presence of RU 1968 100 µM). Because RU 1968 did not interfere with agonist-induced InsP3 formation, the reduction of carbachol-dependent Ca2+ mobilization by RU 1968 might be due to competition of InsP3 binding to the Ca2+ release channel. However, when RU 1968 at 100 µM was added to liver microsomal membranes during incubation with 2.74 nM [3H]InsP3, the amount of bound radioligand was not significantly reduced (245 fmol/mg protein in the solvent control, 231 fmol/mg protein in the presence of RU 1968).



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  		FIG. 8. Agonist-induced generation of InsP3 in LM5 cells is not impaired by RU 1968. The formation of InsP3 in LM5 cells was assessed by a radioligand competition assay. LM5 cells were stimulated by 100 µM carbachol in the absence (solvent) or presence of RU 1968 (100 µM). Preincubation with 100 µM RU 1968 or the solvent was performed for 10 min

Acrosome Reaction in Human Spermatozoa

An essential feature of acrosomal exocytosis is an elevation of [Ca2+]i to promote the fusion between the outer acrosomal membrane and the sperm plasma membrane. At 30 µM, RU 3117 slightly but not significantly (P > 0.05) enhanced the incidence of acrosome-reacted sperm from about 2% in the presence of the solvent to 4.2% ± 0.98% (n = 6 in two independent experiments). At concentrations up to 10 µM, RU 1968 by itself did not elicit acrosome reaction (AR). At higher concentrations, RU 1968 enhanced both basal and ionomycin (5 µM)-stimulated AR (data not shown). In addition, RU 1968 (2 µM) profoundly inhibited the AR induced by 1 µM progesterone and 1 µM PGE1 (Fig. 9). When costimulated with both progesterone and PGE1 (1 µM each), acrosomal exocytosis was observed in 10.3% of live spermatozoa. Preincubation with 2 µM RU 1968 reduced the response by approximately 80% (Fig. 9). The fraction of viable spermatozoa was more than 95% in all experiments and was not decreased by incubations with 1 µM progesterone, 1 µM PGE1, or 2 µM RU 1968. Ionomycin (5 µM) reduced sperm viability within 30 min from 97% in the presence of solvent (0.5% dimethyl sulfoxide) to 72% in the presence of the ionophore. Ionomycin at 5 µM increased the percentage of acrosome-reacted spermatozoa by more than 50%. RU 1968 at 2 µM had no inhibitory effect on ionomycin-elicited AR (data not shown).



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  		FIG. 9. Inhibition of agonist-induced AR by RU 1968. Human sperm suspensions were preincubated for 5 min with either RU 1968 (2 µM, solid bars) or solvent (open bars). A second incubation (30 min) was performed in the absence (solvent) or presence of PGE1 (1 µM) or progesterone (1 µM) or both agonists (1 µM each). The acrosomal status was assesed after staining with FITC-conjugated Pisum sativum agglutinin. The incidence of acrosomal exocytosis was assessed in a population of 200 spermatozoa for each data point. Bars represent the mean ± SEM of 6–9 data points of two independent experiments

DISCUSSION

Despite considerable effort made to define the signaling pathways of progesterone in mammalian spermatozoa, the molecular identity of the extragenomic receptor is still elusive. Because regulation of cation channels via progesterone binding to sigma receptors is evident in a variety of cellular systems, we adressed the question of whether the unique sperm progesterone receptor may be related to sigma receptors formerly described in testis membranes [27, 28]. Bonfils et al. [30] recently characterized two novel steroidal sigma receptor ligands, RU 3117 and RU 1968, that bind to multiple sites in rat testis membranes, including sigma receptors. In the present study, we showed that RU 3117 and RU 1968 inversely interfere with the progesterone-mediated signaling in human spermatozoa. Whereas RU 3117 is a weak agonist at the human sperm progesterone receptor, RU 1968 is a potent inhibitor of sperm activation by both progesterone and PGE1. However, our data provide evidence that the functional effects of the steroidal sigma ligands on human spermatozoa are not mediated by binding to classical sigma receptors. The functional effects of RU 3117 were observed at micromolar agonist concentrations, whereas a nanomolar affinity of RU 3117 to sigma binding sites was observed [30]. In addition, progesterone- and RU 3117-mediated effects were not affected by the high-affinity sigma ligands haloperidol and DTG. Prestimulation of spermatozoa with progesterone, but not with PGE1, completely abolished the rise of [Ca2+]i induced by RU 3117. Thus, there is reason to conclude that RU 3117 activates human spermatozoa by interfering with the extragenomic progesterone receptor independent from sigma receptors.

Although RU 3117 displayed agonistic properties at the nongenomic progesterone receptor, the steroidal sigma receptor ligand RU 1968 antagonized the progesterone-induced Ca2+ entry and the induction of AR. Additionally, RU 1968 blocked the signaling pathway originating from the human sperm PGE receptor with an IC50 in the nanomolar range. To discriminate between a sperm-specific phenomenon and a general inhibitory impact of RU 1968 on phospholipase C-dependent signal transduction pathways, we also examined LM5 cells permanently expressing the Gq/11-coupled m5 muscarinic receptor [31]. Preincubation of LM5 cells with RU 1968 resulted in a concentration-dependent inhibition of carbachol-induced Ca2+ transients. The potency of RU 1968 to inhibit agonist-induced Ca2+ signaling in LM5 cells was in the low micromolar range.

High-affinity binding of RU 1968 to several distinct receptors was deemed to be rather unlikely. A common downstream element of the signal transduction pathways leading to increases in [Ca2+]i may rather represent the target of RU 1968. In accord with such an assumption, carbachol-mediated inositol phosphate turnover was found to be inhibited by sigma receptor ligands in rat brain [23]. Thus, we addressed the issue of whether sigma receptor ligands would inhibit phospholipase C activity. In LM5 cells, however, RU 1968 failed to inhibit agonist-induced InsP3 formation and did not compete with InsP3 for binding to the microsomal InsP3 receptor. Thus, the inhibitory effect of RU 1968 on carbachol-dependent Ca2+ mobilization in LM5 cells might be due to an allosteric block of Ca2+ release via intracellular InsP3 receptors. Along these lines, an allosteric modulation of InsP3-gated ion channels by ATP was described for native [35] and recombinant [36] InsP3 receptors. Furthermore, xestospongins were characterized as cell-permeant blockers of InsP3-mediated Ca2+ release without interacting with the InsP3 effector site [37].

The mechanism by which RU 1968 inhibits agonist-induced Ca2+ entry pathways in human spermatozoa is still unclear. However, sigma receptors do not appear to be involved. In light of the situation that several independent signaling pathways form a redundant system for sperm activation, one must ensure that drugs aimed at compromising sperm function efficiently suppress several independent signaling pathways. RU 1968 is the first steroidal antagonist that potently blocks both progesterone- and PGE1-dependent signaling events in human spermatozoa. The preferential block of signal transduction pathways in spermatozoa supports the development of more selective and effective drugs structurally related to RU 1968 to target the male gamete for pharmacological contraception.

ACKNOWLEDGMENTS

We thank Nadine Albrecht and Eckhard Friedreich for technical assistance. LM5 cells were donated by Lutz Birnbaumer (Los Angeles, CA).

FOOTNOTES

First decision: 3 November 1999.

1 This work was supported by grants from Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, the Deutsche Forschungsgemeinschaft, and the Fonds der Chemischen Industrie. Back

2 Correspondence: Thomas Gudermann, Institut für Pharmakologie, Freie Universität Berlin, Thielallee 67-73, D-14195 Berlin, Germany. FAX: 49 30 8445 1818; guderman{at}zedat.fu-berlin.de Back

3 Current address: Institut für Pharmakologie und Toxikologie, Philipps-Universität Marburg, Fachbereich Humanmedizin, Karl-von-Frisch-Str. 1, 35033 Marburg, Germany. Back

Accepted: February 8, 2000.

Received: October 15, 1999.

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