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Originally published In Press as doi:10.1074/jbc.M403913200 on July 22, 2004

J. Biol. Chem., Vol. 279, Issue 38, 40194-40203, September 17, 2004
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Particulate Adenylate Cyclase Plays a Key Role in Human Sperm Olfactory Receptor-mediated Chemotaxis*

Marc Spehr{ddagger}§, Katlen Schwane¶, Jeffrey A. Riffell||, Jon Barbour¶, Richard K. Zimmer||**, Eva M. Neuhaus¶, and Hanns Hatt¶

From the {ddagger}Department of Anatomy and Neurobiology, University of Maryland, Baltimore, Maryland 21201, the Lst. Zellphysiologie, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany, the ||Department of Ecology and Evolutionary Biology, University of California, Los Angeles, California 90095-1606, and the **Department of Neurosciences, Brain Research Institute, University of California, Los Angeles, California 90095-1606

Received for publication, April 8, 2004 , and in revised form, July 6, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human sperm chemotaxis is a critical component of the fertilization process, but the molecular basis for this behavior remains unclear. Recent evidence shows that chemotactic responses depend on activation of the sperm olfactory receptor, hOR17-4. Certain floral scents, including bourgeonal, activate hOR17-4, trigger pronounced Ca2+ fluxes, and evoke chemotaxis. Here, we provide evidence that hOR17-4 activation is coupled to a cAMP-mediated signaling cascade. Multidimensional protein identification technology was used to identify potential components of a G-protein-coupled cAMP transduction pathway in human sperm. These products included various membrane-associated adenylate cyclase (mAC) isoforms and the Golf-subunit. Using immunocytochemistry, specific mAC isoforms were localized to particular cell regions. Whereas mAC III occurred in the sperm head and midpiece, mAC VIII was distributed predominantly in the flagellum. In contrast, Golf was found mostly in the flagellum and midpiece. The observed spatial distribution patterns largely correspond to the spatiotemporal character of hOR17-4-induced Ca2+ changes. Behavioral and Ca2+ signaling responses of human sperm to bourgeonal were bioassayed in the presence, or absence, of the adenylate cyclase antagonist SQ22536. This specific agent inhibits particulate AC, but not soluble AC, activation. Upon incubation with SQ22536, cells ceased to exhibit Ca2+ signaling, chemotaxis, and hyperactivation (faster swim speed and flagellar beat rate) in response to bourgeonal. Particulate AC is therefore required for induction of hOR17-4-mediated human sperm behavior and represents a promising target for future design of contraceptive drugs.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Despite a century of research, fertilization remains one of the least understood fundamental biological processes (1). Chemical communication between sperm and egg is critical in sexual reproduction, yet the contribution of soluble egg factors is still unclear. Sperm activation and chemotaxis have been demonstrated in marine broadcast spawners, as well as in terrestrial animals (including humans) with internal fertilization (2-6). Chemically mediated behavior thus is a key component of sperm-egg dynamics, in environments ranging from the turbulent ocean to the relatively benign mammalian reproductive tract.

Olfactory receptor proteins (ORs),1 classic G-protein-coupled receptors, comprise the largest gene family in the human genome (7). They are usually, but not always, expressed in the ciliary compartments of nasal olfactory sensory neurons and are coupled to complex signal transduction pathways. Activation of these pathways leads to stimulation of mAC III and finally an increase in intracellular Ca2+ and Na+ concentrations inducing membrane depolarization (8). Interestingly, several ORs are expressed predominantly or exclusively in human sperm cells (9-11). We recently reported chemotaxis by human sperm in response to floral scents (e.g. bourgeonal). The Ca2+ influx mediated by bourgeonal binding to an identified receptor protein (hOR17-4) modified flagellar beating, thus directing cell motility (12). Unresolved, however, is the mechanistic basis for the transduction events that lead from odor signals to swimming responses.

In sperm, hOR17-4-mediated changes in both intracellular Ca2+ and swimming behavior appear to depend on a cAMP-regulated pathway (12). However, the identities and properties of the AC(s) involved in mammalian, especially human, spermatozoa are still debated. All nine mAC isoforms identified in somatic mammalian cells convert the cellular energy carrier ATP into the second messenger cAMP using a specific adenosine binding site (P-site) at the heterodimeric active center of the enzyme. Alternatively, an unconventional soluble adenylate cyclase (sAC), identified in sperm of some mammals (13), shows a decidedly divergent catalytic domain (14). Thus, there is evidence for both a G-protein-activated mAC(s) in mouse sperm (15-17) and an exclusive or predominant role of sAC in spermatozoa of other mammals (13, 18, 19). This bicarbonate and Ca2+-activated sAC is structurally related to prokaryotic cyclases but is insensitive to G-proteins or P-site ligands.

The goal of this study was to determine the molecular mechanisms that couple OR activation to changes in human sperm swimming behavior. Specifically, we analyzed the expression, spatial distribution, and involvement in cellular and behavioral function of distinct signaling proteins (hOR17-4, G-proteins, and mACs). These studies indicate that, similar to nasal sensory neurons, activation of mAC(s) plays a key role in sperm OR-dependent signaling processes. Specifically, our results implicate mAC(s), rather than sAC, as a molecular link between OR activation and sperm behaviors such as chemotaxis (directed movement with respect to a chemical gradient), swimming (elevated speed), and asymmetric flagellar beating (hyperactivation).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sperm Preparation—Human sperm were freshly obtained from young healthy donors. For Ca2+ imaging, acrosome reaction assays, and immunocytochemistry a Percoll density gradient centrifugation was performed after liquefaction (30 min at 35.5 °C) to isolate mature and motile sperm (20). In brief, liquefied semen was overlaid on a two-layer Percoll (Amersham Biosciences) density gradient consisting of 80 and 55% isotonic Percoll in Ham's F-10 medium (Invitrogen). After 40-min centrifugation at 500 x g at room temperature the pellet was collected, washed in standard Ringer solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM Hepes, 10 mM glucose), and again centrifuged for 15 min.

For MudPIT protein expression analysis, pooled human semen samples were snap-frozen by quenching in liquid N2 and stored at -70 °C until required. 50 ml of pooled ejaculate was thawed on ice, and all subsequent steps were performed at 4 °C unless otherwise stated. Spermatozoa were enriched as described above and resuspended in standard Ringer's solution with protease inhibitors (Roche Complete® protease inhibitor mixture). Cells were lysed by homogenization (Ultraturax; 1200 units/min for 1 min) and subsequent sonification for 10 s at 40 watts (Sonifier B12, Branson Sonic Power Co.). Cell debris and nuclei were removed by centrifugation (1,000 x g, 10 min), the pellet was discarded, and the supernatant was centrifuged for 2 h at 100,000 x g. The resultant membrane pellet was solubilized using 1% (w/v) L-{alpha}-lysophosphatidylcholine (Sigma); protein concentration was determined by using the Bradford microassay (Bio-Rad).

For bioassays frozen sperm of healthy donors was obtained from California Cryobank (Los Angeles, CA) and prepared according to World Health Organization guidelines (21). Each sperm suspension was brought to 37 °C and adjusted to 106 cells/ml in bioassay chamber wells (see under "Sperm Motility Analysis").

Expression Analysis via MudPIT, SDS-PAGE, and In-gel Digestion—A sample aliquot (100 µg) in Laemmli buffer (30% glycerol, 3% SDS, 125 mM Tris/Cl, pH 6.8) was resolved by 8% SDS-PAGE. Coomassie-stained bands were excised, pooled, and washed thrice in 100 mM NH4HCO3 (pH 8.5) in 50% acetonitrile (ACN) for 20 min. Colorless gel pieces were washed in 100% ACN for 10 min and dried by vacuum centrifugation for 15 min at room temperature. Gel pieces were reduced with 20 mM dithiothreitol in 100 mM NH4HCO3, 5% acetonitrile for 1 h at 55 °C, washed (100 mM NH4HCO3 for 10 min), dehydrated (100% ACN 20 min), and alkylated (100 mM iodoacetamide/100 mM NH4HCO3 for 30 min (dark/room temperature). After further washing, dehydration, and drying, the pieces were rehydrated in digestion buffer (40 mM ammonium bicarbonate/9% ACN containing proteomics grade trypsin (Sigma) at an enzyme:substrate ratio of 1:50) and incubated overnight at 37 °C. Peptides were extracted using 1% trifluoroacetic acid in 50% acetonitrile for 20 min. The gel pieces were vortexed for 5 min before recovering the supernatant. The extracted peptides were concentrated by vacuum centrifugation and subjected to LC/LC/MS/MS.

LC/LC/MS/MS—A fused-silica microcapillary column was laser-pulled (Model P-2000, Sutter Instrument Co., Novato, CA) and successively packed with 10 cm of 5-µm C18 reverse-phase material (XDBC18, Hewlett Packard) and 4 cm of 5-µm strong cation exchange material (SCX-Partisphere, Whatman, Clifton, NJ) as previously described (22). The sample was bomb-loaded and analyzed using fully automated online LC/LC/MS/MS instrumentation (HPLC LC Packings Ultimate, MS, Finnigan LTQ). The resultant MS spectra were searched against the Swiss-Prot data base of Homo sapiens using SEQUEST.

Immunocytochemistry—The following primary antibodies were used: (a) a rabbit polyclonal antibody against mAC III (Santa Cruz Biotechnology Inc., Santa Cruz, CA); (b) a goat polyclonal antibody against mAC VIII (Santa Cruz Biotechnology); and (c) a rabbit polyclonal antibody against Golf (generous gift from Dr. R. R. Reed). For immunofluorescence secondary goat anti-rabbit or donkey anti-goat conjugated to Alexa 488 or Alexa 568 from Molecular Probes were used.

Enriched spermatozoa were plated on polylysine-coated coverslips (15 min/room temperature). Coverslips were washed in Ringer solution and fixed (30 min/room temperature) in 3% paraformaldehyde in Ringer solution containing 10 mM glucose. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 in PBS containing 0.5% cold water fish skin gelatin (Sigma) (15 min/room temperature). Cells were incubated with primary antibodies diluted 1:500 in PBS/gelatin/Triton X-100 for 45 min, washed, and incubated with fluorescently labeled secondary antibodies diluted 1:500 in PBS/gelatin/Triton X-100 for 45 min; nuclei were stained with DAPI (Molecular Probes) in this incubation step. Unbound antibodies were washed with PBS, and coverslips were mounted in ProLong Antifade (Molecular Probes).

All fluorescence images were obtained with a confocal microscope (LSM510 Meta; Zeiss) using a 40x 1.4-numerical aperture objective (pinhole set to one Airey unit) and further processed with Photoshop (Adobe Systems Inc., San Jose, CA).

Acrosome Reaction Assay—Acrosome-reacted sperm were visualized according to Meizel and Turner (23), incubating 100 µl of sperm suspension at a time with 5 µl of fluorescein isothiocyanate-conjugated peanut lectin (Sigma). Acrosome-reacted cells were plated on concanavalin A (Sigma)-coated 35-mm dishes and counted using a digital Zeiss fluorescence microscope.

Single Cell Ca2+ Imaging—Cell density was photometrically adjusted to an extinction of E260 nm = 0.035. Sperm were incubated (45 min/35.5 °C) in loading buffer (pH 7.4) containing Ringer solution and 7.5 µM fura-2-AM (Molecular Probes), and 0.1% Pluronic F-127 (Sigma). Next, sperm was centrifuged at 500 x g for 15 min, and the pellet was resuspended in fura-2-free buffer solution. 100 µl of mature motile fura-2-AM-loaded spermatozoa was transferred to concanavalin A (Sigma)-coated 35-mm dishes (30 min at 35.5 °C).

Ca2+ imaging was performed using a Zeiss inverted microscope equipped for ratiometric imaging (24). Ca2+ images were acquired from up to 15 spermatozoa in a randomly selected field of view, and integrated fluorescence ratios (f340/f380 ratio) were measured by separating the sperm head and midpiece. Exposure to bourgeonal (Quest Int., Naarden, Netherlands) and/or the water-soluble mAC inhibitor SQ22536 (Calbiochem) was accomplished using a specialized microcapillary application system (24). Only spermatozoa with their heads and midpiece attached and their tails beating were included in the analysis.

Sperm Population Ca2+ Screening—Human fura-2AM-loaded sperm was diluted in Ringer solution (cell density adjusted to E260 = 0.035) and transferred to a 96-well Optiplate (PerkinElmer Life Sciences) with 150 µl of suspension per well. After addition of different test solutions (normal Ringer's solution (negative control), SQ22536, bourgeonal, progesterone (positive control)) samples were immediately read with a Fusion-{alpha} Universal Multiplate Analyzer (PerkinElmer Life Sciences) using a dual sequential fluorescence mode. Wells were subsequently illuminated (1 s) using a filter of 330 ± 20 nm and a filter of 380 ± 10 nm. Fluorescence signals were detected using a filter of 515 ± 10 nm, and ratios were calculated. Fluorescence changes due to Ringer solution application were subtracted from the originally recorded signals.

Sperm Motility Analysis—Microcapillary bioassays were combined with computer-assisted video motion analysis (CAVMA) to determine behavioral effects of mAC signaling. Four replicate trials were performed for each of 12 treatments in a 6 (incubations) x 2 (stimuli) factor design. Sperm from different human donors were used in each trial. Experiments were conducted in compartments with two wells connected by a channel. First, sperm suspensions were incubated for 30-40 min in a pair of wells containing one of six treatments: HEPES-buffered human tubal fluid alone (HTF control, Irvine Scientific, Santa Ana, CA) or with addition of 10, 5, 1, 0.1, or 0.01 mM SQ22536. Second, to each treatment, a 6-µl, flat, microcapillary tube was inserted into the channel with the ends bridging sperm suspensions (106 cells/ml) in opposing wells. The stimulus (HTF medium or 10-7 M bourgeonal) was presented in a capillary. After 1-h incubation at 37 °C, the contents of each capillary was transferred to the well of a toxiplasmosis slide, heat-fixed, and stained with 0.1% acridine orange for 1 min. The number of cells was then counted using an Olympus model BH2 microscope (x67 magnification) equipped for phase-contrast and epifluorescence applications.

Swimming speeds and directions of individual cells were videotaped over 15 s, beginning 10-15 min after an assay began. Integrating with respect to time, fluid dynamic theory predicts that the concentration gradient created by continuous bourgeonal release should reach steady state in ~10 min, within 300 µm of the capillary tip (12, 25). Sperm images were recorded on magnetic tape using a cooled charge-coupled device camera (NEC model TI 23A) mounted on an inverted compound light microscope (Olympus IX70, x90 magnification) (26). The camera had a 100-µm depth of field and focused on a region within 300 µm of the tip. Fluid dynamic theory further predicts that drag forces have especially strong effects on flagellar motion within ~10 sperm body lengths of a wall, or microscope slide surface. To minimize potential artifacts, we assayed sperm motility in response to chemical stimuli at ~2 mm, or 70 sperm body lengths, from the nearest chamber wall (26).

Images were digitized at 30 frames/s and processed using a CAVMA system (Motion Analysis Corp., Model VP320) interfaced with a Sun SPARC2 computer workstation (12). To avoid parallax, we discarded short paths (≤10 frames) in which apparent sperm size changed >20%. All other paths were included in analyses. Swimming speed of each individual sperm was determined on a frame-by-frame basis and averaged over each path. The angle of sperm orientation was measured with respect to an origin (0°), defined as the shortest tangent between each cell and the capillary tip. Using circular statistics, a mean vector (r) was calculated to describe the average direction of movement of a cell population. Whereas a vector length of one indicates that all cells swim in a single, common direction, a vector length of zero indicates random motion. To determine if cell movement within a chemical gradient was non-random, each mean direction was compared with a uniform circular distribution using a Rayleigh's test.

The production of a bourgeonal concentration gradient was vital to the success of this experimental design. Fluorescent (rhodamine) dye, as a surrogate for bourgeonal, was released from the capillary tip. Planar laser-induced fluorescence imaging revealed a steep, ascending gradient in dye concentration. Rhodamine concentration (a) remained highest at the tip, decreasing as approximately the three-halves power of time, and (b) decreased geometrically as a function of 1/distance from the tip. After 10 min, for example, dye concentration was reduced 10- and 100-fold within 15 and 180 µm of the capillary tip, respectively. In addition, using non-motile sperm as tracers for flow visualization and CAVMA, fluid motion (due to convection, flow from pipette, and mixing) was negligible (<5 µm/s) as compared with the swimming speeds of live cells.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of hOR17-4, Stimulatory G-proteins, and mAC(s)—Initially, we asked whether potential signaling proteins that could link sperm OR activation to cAMP-mediated behavioral changes, such as mAC isoforms and stimulatory G-protein {alpha} subunits, are expressed in human sperm cells. Because mature mammalian spermatozoa have stopped protein synthesis (27), this analysis cannot be performed at the mRNA level (e.g. by reverse transcription-PCR or in situ hybridization). Therefore, we decided to introduce a state-of-the-art "shotgun"-proteomics approach (MudPIT) to the analysis of sperm protein content that is based on two-dimensional liquid chromatography and mass spectroscopy (22).

Sperm membrane proteins were separated using one-dimensional gels and subjected to tryptic digestion. All peptide MS/MS spectra were searched using the SEQUEST search criteria allowing for variable modification of methionine (+16 Da) and cysteine (+57 Da). Spectra were assigned as good hits according to Wolters et al. (28) with all good candidates being manually evaluated. Among ~100 different membrane proteins detected, this approach identified a set of candidate molecules (Table I), including all nine mammalian mAC isoforms and the "olfactory-specific" stimulatory Golf {alpha}-subunit (29). Furthermore, MudPIT analysis confirms expression of hOR17-4 in human sperm. Thus, several components necessary for G-protein-coupled cAMP generation are present in human spermatozoa.


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  		TABLE I
MudPIT reveals expression of candidate OR-associated signaling proteins in human sperm

 
Signaling Components Show Distinct Spatial Distribution—Given an OR-mediated signaling pathway, we next determined the spatial distribution of the "olfactory-specific" Golf and mAC III as well as the prominent mAC VIII isoforms. Isolated human spermatozoa were immunostained with anti-mAC III, anti-mAC VIII, and anti-Golf antibodies to examine the (sub-) cellular distributions of the proteins. Spermatozoa were clearly labeled by all of these antibodies (Fig. 1, left panel), whereas control samples labeled with the same secondary antibodies alone lacked any significant staining (Fig. 1, right panel). Interestingly, localization of mAC isoforms differed between different compartments: mAC VIII was strongly localized to the sperm tail, whereas little or no staining was observed on the head and on the midpiece (Fig. 1b). By contrast, mAC III localization differed from cell to cell; some spermatozoa showed a relatively uniform distribution of the protein on the head, although in varying intensities (Fig. 1a, arrows). Other cells showed a ring-like staining around the head (Fig. 1a, arrowhead), which we suspect reflects the equatorial segment. We next investigated the dependence of these localization differences on the acrosome status of the cells and found that strong uniform staining only occurs in acrosome-reacted cells (Fig. 2a). In agreement with previous reports (4), only 6.36 ± 1.57% of more than 2500 sperm cells investigated (5 donors and 15 different samples) were found spontaneously acrosome-reacted, and this ratio was not significantly changed by 10-min incubation with bourgeonal (50 µM) and/or the water-soluble P-site inhibitor of mAC function SQ22536 (3 mM) (Fig. 2b). Most spermatozoa show a relatively strong middle piece staining with antibodies against mAC III, and the staining of the tail in most cases was relatively faint (Fig. 1b). Golf labeling was observed throughout the tail as well as in the middle piece of the spermatozoa (Fig. 1c). Thus, both Golf and mAC III/VIII are localized to the sperm midpiece and/or tail where flagellar beating is initiated.



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  		FIG. 1.
Distribution of signal transduction components in spermatozoa. a, an antibody directed against the mAC III labeled the head and the midpiece to varying extents, some cells show staining of the head to various extents (arrows), some show a ring-like staining around the head (arrowhead). The midpiece is also stained by the antibody, although not in all cells very prominently. Some fainter staining is present in the tail. b, anti-mAC VIII antibodies showed strong labeling of the tail, with little to no staining of head and midpiece. c, Golf is present in the midpiece and the tail of human spermatozoa. Primary antibodies are detected with A488-labeled secondary antibodies and shown in green, and nuclei are counterstained with DAPI (blue). The right control panel contains representative images of samples labeled with the corresponding secondary antibodies alone under exactly the same experimental conditions. Single confocal sections. Bar, 10 µm.

 



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  		FIG. 2.
AC distribution is dependent on the acrosome status. a, spermatozoa preparations were incubated with an A568 dye conjugate of lectin peanut agglutinin, which can be used as selective marker for human sperm acrosomes (red), stained with anti-mAC III antibodies (green), and nuclei are counterstained with DAPI (blue). Overlay image demonstrates strong staining of the whole head of acrosome-reacted spermatozoa with mAC III, whereas spermatozoa, which have not undergone acrosome reaction (no red staining), show typical ring-like staining around the head. Single confocal sections. Bar, 10 µm. b, the proportion of spontaneously acrosome-reacted sperm is low under control conditions and not significantly changed by preincubation with bourgeonal (50 µM) and/or SQ22536 (3 mM).

 
Odorant-induced Ca2+ Signals Originate in the Flagellar Midpiece—Generation of odorant-induced Ca2+ signals in sperm cell compartments (12) should correspond with the functional localization of hOR17-4 and its signal transduction machinery. Therefore, we investigated the spatiotemporal properties of the bourgeonal response of individual spermatozoa using high resolution Ca2+ imaging. In this experiment, stimulus-induced Ca2+ signals were analyzed separately in the head and flagellar midpiece. A relatively high temporal resolution (of four complete fluoratiometric recording cycles per second) allowed for a sub-cellular analysis of the bourgeonal-induced Ca2+ response (Fig. 3).



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  		FIG. 3.
Ca2+ signals originate in the flagellar midpiece. Representative separate ratiofluorometric recordings of both the attached head and midpiece of an individual fura-2-loaded human spermatozoon. The cytosolic Ca2+ level is depicted as the integrated fluorescence ratio (f340/f380) and viewed as a function of time. A 12-s bourgeonal stimulation induced instantaneous Ca2+ responses in the midpiece, whereas response time was remarkably delayed in the head region (dashed lines indicate the respective onset of the Ca2+ signal). From a total of 17 individual recordings, an average delay of 2.15 ± 1.2 s between the onset of the signals in midpiece and head can be reported. Inset shows a fluorescence image of a fura-2-loaded spermatozoon with head and midpiece marked as independent regions of interest.

 
In 85% of all cells individually tested (17/21) the initial response to bourgeonal was observed in the midpiece region. With a mean latency of 2.15 ± 1.2 s, Ca2+ increases were detectable in the sperm head. Although the overall response waveform was comparable in both compartments, absolute signals were considerably stronger and more persistent in the head region (38.8 ± 8.7 s) than in the midpiece (26.95 ± 3.8 s). Under Ca2+-free extracellular conditions no signals were detected, in either the head or the midpiece (data not shown). In summary, these data are consistent with the spatial distribution of potential signaling components shown in our immunolabeling experiments.

Odorant-induced Ca2+ Signaling in Human Sperm Is Mediated via mAC Activation—Having identified several molecules of a potential OR-coupled cAMP signaling system in human sperm cells, we next assessed whether these molecules are used for odorant-induced signaling in response to OR activation. We were particularly interested in the question whether a soluble or a particulate AC is involved in this response. All of the nine mAC isoforms cloned thus far are affected by P-site inhibitors (30), whereas sAC is not inhibited by this class of pharmacological agents (16). It is therefore possible to distinguish between mACs and sACs by using a specific P-site adenylate cyclase antagonist, SQ22536, which selectively blocks mAC but not sAC isoforms.

fura-2-loaded sperm samples were tested for hOR17-4 activation by bourgeonal (500 µM) in the absence or presence of SQ22536 at various concentrations. Stimulation with bourgeonal alone induced robust dose-dependent Ca2+ increases in all human sperm samples tested (n = 17), whereas pure mechanical stimulation was without effect. The average bourgeonal signal was 26% (50 µM) or 54% (500 µM) of the Ca2+ response caused by progesterone (30 µM; Fig. 4a), an important component of human follicular fluid that is known to induce sperm acrosome reaction via indirect opening of different membrane Ca2+ channels (31). This progesterone-induced control signal was unaffected by 90-s preincubation with 3 mM SQ22536 (n = 10; Fig. 4a) suggesting no crucial role of mAC in sperm progesterone signaling cascades. In sharp contrast, pre-incubation with SQ22536 inhibited bourgeonal responses in a dose-dependent manner (Fig. 4b). The dose that produced a half-maximal response (IC50) was ~2 mM. Although previously reported IC50 values in other cell types, e.g. olfactory receptor neurons (32), are considerably lower (~300 µM), inhibition of mammalian sperm enzymes commonly requires increased dosage of pharmacological agents (33). We also investigated the effect of mAC inhibition on individual spermatozoa in single cell Ca2+ imaging experiments (Fig. 4c); in 71% (17/24) of all bourgeonal-sensitive cells (3-s application; 50 µM) 3 mM SQ22536 considerably reduced responses and 10 mM SQ22536 completely blocked bourgeonal signaling. Furthermore, we noted that SQ22536 preincubation (10 mM), in most spermatozoa, produced a slow but reversible decrease in the resting intracellular Ca2+ concentration (Fig. 4c). This indicates some tonic (basal) activity of mAC at rest, in the absence of receptor stimulation. In contrast to particulate AC, the unconventional sAC isoform in sperm has been reported to function as a bicarbonate sensor (13, 14) increasing cytosolic cAMP in response to millimolar bicarbonate concentrations (EC50 ~ 25 mM). In another set of single cell Ca2+ imaging recordings, we specifically activated sAC by applying short (3 s) pulses of bicarbonate (100 mM; Fig. 4d). In 169 cells investigated, the resulting Ca2+ responses showed no sign of inhibition by SQ22536 (3 mM). The average Ca2+ response amplitude in presence of SQ22536 was 105% compared with the initial bicarbonate response (data not shown), demonstrating that sAC is not targeted by this pharmacological agent even at high doses. Given the lack of effects of 3 mM SQ22536 on progesterone- and bicarbonate-induced Ca2+ responses, our data strongly indicate the specific inhibition of mAC(s) via SQ22536. Taken together, the above findings suggest a key role for mAC(s) in sperm hOR17-4 signaling.



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  		FIG. 4.
Sperm hOR17-4-mediated Ca2+ signaling is coupled to mAC activation. a, fura-2-loaded sperm suspensions respond to bourgeonal in a dose-dependent manner (average fluoratiometric Ca2+ signal amplitudes are normalized to the mean progesterone response). Progesterone-mediated Ca2+ signals are not affected by SQ22536 (3 mM), a specific P-site blocker of mACs. b, depending on the co-applied concentration, SQ22536 (0.1-10 mM) inhibits sperm suspension bourgeonal (500 µM) sensitivity, with an IC50 of ~2 mM. c, single cell Ca2+ imaging confirms population screenings. A representative fluoratiometric recording of an individual fura-2-loaded human spermatozoon. The cytosolic Ca2+ level is depicted as the integrated fluorescence ratio (f340/f380) and viewed as a function of time. 3-s bourgeonal (50 µM) pulses (*) induce transient Ca2+ signals that can be partly (3 mM) or completely (10 mM) inhibited by SQ22536 preincubation (70 s). d, using the same experimental protocol, bicarbonate (*)-mediated (100 mM) Ca2+ signals in single cells remain unaffected by SQ22536 (3 mM).

 
Sperm Behavioral Responses to Bourgeonal Require mAC Function—Human sperm express marked behavioral responses to bourgeonal. At concentrations of ≥10-7 M, cells accumulate at significantly higher numbers in microcapillary tubes that present bourgeonal in an ascending, as opposed to a uniform or descending concentration gradient (12). Moreover, human sperm exhibit chemotaxis, orienting strongly within an ascending gradient. Here, microcapillary bioassays were combined with CAVMA to determine effects of mAC signaling on bourgeonal-stimulated behavior. Sperm swimming velocities and trajectories were quantified in a series of assays, where microcapillaries of either human tubal fluid (HTF) (control medium) or bourgeonal (known stimulant) were added to cells incubated in either HTF or the adenylate cyclase inhibitor, SQ22536. Gamete response to an ascending gradient of 10-7 M bourgeonal was as expected: swim speeds, directed movements, rates of flagellar beating, and cell densities in capillaries increased relative to the HTF control (Figs. 5 and 6). Sperm, swimming toward the microcapillary tip, sensed an increasing gradient, beat their flagella symmetrically, and arrived at the terminus without redirection (Fig. 6). Alternatively, sperm initially swimming away from the capillary abruptly turned, via an asymmetrical flagellar beat, to reorient toward the tip (Fig. 7). Sperm were thus also hyperactivated by bourgeonal and ultimately navigated along an ascending gradient via chemotaxis.



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  		FIG. 5.
Swimming behavior of human sperm imaged near a microcapillary tip and processed using CAVMA. While being incubated in either SQ22536 (0.1, 1, 5, or 10 mM) or HTF, sperm were challenged with an ascending gradient of bourgeonal (a) or exposed to human tubal fluid (HTF) as control (b). Open circles correspond to video images captured at intervals of 0.033 s; arrowheads indicate directions of travel for individual cells. A minimum of 50 paths was analyzed for each test or control solution. To eliminate selection bias, we used a random number generator to choose representative paths from the entire pool. However, complete data sets, not representative paths, were used to calculate mean (±S.E.) swimming speeds (w), mean angles ({theta}), and mean vector lengths (r). The angle of sperm orientation was measured with respect to an origin (0°), here defined as the shortest tangent between each individual cell and the capillary tip. To determine if cell movement within a chemical gradient was significant (P), we used Rayleigh's test, comparing each mean direction against a uniform circular distribution.

 



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  		FIG. 6.
Effects of P-site inhibitor (SQ22536) on sperm behavioral responses to bourgeonal and human tubal fluid (HTF, control). a, accumulation of sperm in microcapillary tubes. b, flagellar beat frequency of cells swimming ≤ 100 µm of the capillary tip. While being incubated in either SQ22536 (0.1, 1, 5, or 10 mM) or HTF, sperm were challenged with an ascending gradient of bourgeonal, or exposed to human tubal fluid as control. An asterisk indicates a significant difference between test and control stimuli for each incubation treatment (one-way ANOVA, followed by Scheffe test: *, p < 0.05; ***, p < 0.001). Each plotted value is a mean (±S.E.).

 



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  		FIG. 7.
Analysis of bourgeonal-induced motility patterns. a, swimming behavior of human sperm imaged near a microcapillary tip and processed using CAVMA. Top, left: a representative path demonstrating a chemotactic "flip turn" by sperm having detected a decrease in bourgeonal concentration while swimming away from the terminus. Open and closed circles correspond to video images captured at intervals of 0.033 s; arrowheads indicate direction of travel for an individual cell. Top, right: seven sequential frames of digital video images corresponding to closed circles in the path to the left. Highlighted are three video frames that specifically show the asymmetrical flagellar bending that occurs during a single chemotactic turn. Middle: sperm fail to exhibit a chemotactic turn in response to a decrease in bourgeonal concentration, when cells are incubated in 10 mM SQ22536. Bottom panel: similarly, sperm swim without rapid turns when challenged with HTF (control), as opposed to bourgeonal. b and c, mean (±S.E.) percentages of total sperm populations entering, or leaving, respectively, microcapillary tubes over 30-s video recording intervals (see text for details). Cells were included in the analysis only if they were initially swimming ≤100 µm of the tip. d, of those sperm leaving microcapillary tubes, mean (±S.E.) percentages of cell populations exhibiting chemotactic flip turns and re-entering the termini. For b-d, an asterisk indicates a significant difference between test and control stimuli for each incubation treatment (one-way ANOVA using arsine transformed data, followed by a Scheffe test: *, p < 0.05).

 
In contrast, when 10-7 M bourgeonal capillaries were placed in 5 or 10 mM SQ22536 incubations, sperm behavior was indistinguishable from that in the HTF control (Figs. 5 and 6). Moreover, in the absence of bourgeonal, SQ22536 had no measurable effect on gamete movements. Thus, inhibition of bourgeonal-induced hyperactivation and navigation was specifically and unequivocally associated with SQ22536.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study provides several lines of evidence for the physiological significance of mAC signaling in navigating human sperm. These findings give new insights into the initial steps that transduce chemodetection into ordered changes in swimming behavior.

Previously, evidence for hOR17-4 expression in sperm was limited to functional assays (12), primarily due to the lack of reliable antibodies against human odorant receptors. Here, we overcame this limitation by introducing MudPIT analysis to the investigation of sperm proteins. Our results confirm hOR17-4 expression, thereby contributing important new evidence for the role of the receptor in human sperm behavior.

In addition to hOR17-4, a set of stimulatory G-proteins, the mAC VIII isoform (not all of which have yet been linked to OR activation in vivo) and the "olfactory-specific" G{alpha}olf and mAC III proteins (29, 34) were also identified in human sperm cells. In the central nervous system the highest expression of mAC III is exhibited in the olfactory neuroepithelium, whereas the important site of mAC VIII expression is the hypothalamus (35). G{alpha}olf has the capacity to stimulate both mAC III and mAC VIII (36, 37), whereas both enzymes are unaffected by G{alpha}i or G{beta}{gamma} (38). Increased Ca2+ concentration has the opposite effects on mAC isoforms III and VIII. Although Ca2+/calmodulin complexes stimulate mAC VIII (39), calmodulin kinase II activity is reported to inhibit mAC III (40). In mouse germ cells and spermatozoa mAC III and/or mAC VIII expression has also been reported (18, 41, 42). Although mice deficient in either mAC VIII or G{alpha}olf are fertile but rarely surviving (43, 44), homozygous matings of mAC III K.O. mice were unproductive, although they showed normal mounting and anogenital sniffing behaviors (45). However, it remains to be determined if OR-mediated sperm signaling pathways uncovered in humans can be transferred to the mouse model, because fertilization barriers between species may also be reflected on the molecular level. Noteworthy, physiological bourgeonal concentrations do not elicit any detectable Ca2+ signals in mouse sperm,2 precluding investigation in a genetically altered mouse model.

Subregional analysis of bourgeonal-induced Ca2+ signals in individual sperm suggests restriction of the early responsive components to the flagellar midpiece. High resolution imaging shows that activation of the Ca2+ pathway invariably spreads from the tail to the head region. This spatiotemporal response character reflects putative localization of hOR17-4 on the midpiece, the mitochondria-rich structure of the flagellum that provides the energy required for extensive beating (46). Localization of ORs on the midpiece has also been reported for dog sperm (11). For human spermatozoa, we additionally demonstrate partial co-localization of G{alpha}olf and mAC III by immunocytochemistry, favoring their potential participation in OR-mediated signal transduction. Moreover, spatial distribution of mAC III in the sperm head appears dependent on the acrosome status of the cell, showing restricted localization to the presumable equatorial segment in non-acrosome-reacted sperm. Although treatment of sperm with bourgeonal and/or SQ22536 does not significantly change the proportion of acrosome-reacted cells, thus suggesting no relevance of the hOR17-4 signaling pathway in the initiation of the acrosome reaction, the role of mAC III in this essential sperm behavior will be subject to future investigation. In sperm, as well as in somatic cells, cellular signaling is spatially restricted via molecular anchors, scaffolds, or adaptor proteins (47). However, coincidental co-localization in contrast to specific organization in transducisomes cannot be excluded. Technical limitations currently preclude recording Ca2+ signals in beating flagella (see "Experimental Procedures"). Accordingly, we cannot assess potential Ca2+ signals in the sperm tail, the major site of mAC VIII expression. Because tail expression of hOR17-4 cannot be excluded and because mAC VIII as well as G{alpha}olf are co-localized along the whole flagellum, potential OR-mediated signaling in distal tail regions requires further study.

It remains unclear whether the midpiece Ca2+ increase diffuses slowly into the head region or if cAMP acts as a diffusing vector that gates Ca2+ channels preferentially located in the head (48). Moreover, it remains to be determined whether the more persistent Ca2+ signal in the head results from lower Ca2+ extrusion capacity and/or phosphodiesterase activity and if this time course serves a specific physiological function.

Sperm Ca2+ responses to bourgeonal, directed movement of sperm along an ascending bourgeonal gradient, and enhanced swimming speed are completely abolished by pharmacological inhibition of mAC(s) with SQ22536, a substituted adenine derivative that forms a dead-end complex with activated mAC(s) by occupying the P-site of the enzyme (49, 50). Due to its unrelated structure sperm sAC lacks sensitivity to P-site ligands (50), thus SQ22536 allows for a functional dissociation of particulate and soluble AC activity. Given the high effective concentrations of water-soluble SQ22536 in our assays, potential nonspecific effects of this drug are of particular concern. However, neither progesterone-induced Ca2+ responses nor presumably sAC-mediated Ca2+ signaling (bicarbonate stimulation) are affected by SQ22536. These controls aim to exclude nonspecific effects on general sperm Ca2+ signaling pathways and validate mAC as the most likely target of SQ22536. The expression and functional significance of mACs controverts the widespread notion that unconventional sACs are predominant in sperm cAMP signaling (for a recent review see Ref. 51). Both the inhibitory effect of a specific P-site mAC inhibitor and protein identification by MudPIT analysis and sub-cellular immunolocalization strongly support the idea that mACs are key mediators in directed sperm movement. However, a positive feedback loop based on secondary activation of sAC by increased intracellular Ca2+ (14) cannot be ruled out.

In addition to its described role in chemotaxis and chemokinesis (12), bourgeonal had a previously undescribed influence on the turn rate and flagellar beat frequency of sperm cells. Such motility patterns accompanied by asymmetrical flagellar movements are thought to underlie chemotactic movement and chemokinesis in a yet unidentified manner (52). Both increased turn rate and higher flagellar beat frequency also require activation of mAC(s). Thus, local elevations in intracellular cAMP that lead to an opening of Ca2+ entry channels (12) are likely to be responsible for bourgeonal-induced changes in flagellar motion (46).

Previous work on the hOR17-4/bourgeonal signaling system (12) has provided a powerful tool to investigate mechanisms of mammalian sperm chemotaxis. The results of the present study provide an improved understanding of the molecular mechanisms underlying the initial signaling events in OR-mediated sperm behaviors. Our data suggest a cascade model in which hOR17-4 ligand binding activates one or more mACs via a stimulatory G-protein, potentially Golf. The proposed pathway is remarkably similar to olfactory signal transduction. In this regard, it will be interesting to determine if cAMP directly opens Ca2+-permeable ion channels in the sperm membrane (e.g. CatSper, CNG, or TRPC2 channels (53-59)) or acts as an activator of downstream signaling components (e.g. protein kinase A (60)). Disclosing the identity of the Ca2+ entry channel that completes the hOR17-4-activated cascade will be a major goal of coming research. Based on this new study, hOR17-4 and co-localized mACs could become important targets for future pharmacological treatment in infertility or contraception.


   FOOTNOTES
 
* This work was supported by the Alma und Heinrich Vogelsang Stiftung. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. Anatomy and Neurobiology, School of Medicine, University of Maryland, 685 West Baltimore St., Baltimore, MD 21201. Tel.: 410-706-8921; Fax: 410-706-2512; E-mail: mspeh001{at}umaryland.edu.

1 The abbreviations used are: OR, odorant receptor; AC, adenylate cyclase; mAC, membrane-bound adenylate cyclase; sAC, soluble adenylate cyclase; MudPIT, multidimensional protein identification technology; CAVMA, computer-assisted video motion analysis; ACN, acetonitrile; LC, liquid chromatography; MS, mass spectrometry; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; HTF, human tubal fluid. Back

2 M. Spehr, K. Schwane, J. A. Riffell, J. Barbour, R. K. Zimmer, E. M. Neuhaus, and H. Hatt, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Charles Sell for providing bourgeonal; Dirk Wolters for assistance and instructions on MudPIT; Randall R. Reed for providing Golf antibody; Harald Bartel, Meriyem Aktas, and Stefan Köster for assistance; and Frank Zufall, Michael T. Shipley, Kyrill Ukhanov, and Cheryl Ann Zimmer for comments and suggestions on the manuscript.



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