Biol Reprod Keystone Symposia Conference on Frontiers in Reproductive Biology & Regulation of Fertility.
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BOR - Papers in Press, published online ahead of print November 16, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.047837
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BIOLOGY OF REPRODUCTION 74, 492–500 (2006)
DOI: 10.1095/biolreprod.105.047837
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Signaling Pathways for Modulation of Mouse Sperm Motility by Adenosine and Catecholamine Agonists1

Sonya M. Schuh 3, Anne E. Carlson 3, G. Stanley McKnight 4, Marco Conti 5, Bertil Hille 3, and Donner F. Babcock 2 3

Departments of Physiology and Biophysics3 and Pharmacology,4 University of Washington, Seattle, Washington 98195 Department of Gynecology and Obstetrics,5 Division of Reproductive Biology, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT

Capacitation of mammalian sperm, including alterations in flagellar motility, is presumably modulated by chemical signals encountered in the female reproductive tract. This work investigates signaling pathways for adenosine and catecholamine agonists that stimulate sperm kinetic activity. We show that 2-chloro-2'-deoxyadenosine and isoproterenol robustly accelerate flagellar beat frequency with EC50s near 10 and 0.05 µM, respectively. The several-fold acceleration is maximal by 60 sec. Although extracellular Ca2+ is required for agonist action on the flagellar beat, agonist treatment does not elevate sperm cytosolic [Ca2+] but does increase cAMP content. Acceleration does not require the conventional transmembrane adenylyl cyclase ADCY3, since it persists in sperm of ADCY3 knockout mice and in wild-type sperm in the presence of the inhibitors of conventional adenylyl cyclases SQ-22536, MDL-12330A, or 2', 5'-dideoxyadenosine. In contrast, the acceleration by these agents is absent in sperm that lack the predominant atypical adenylyl cyclase, SACY. Responses to these agonists are also absent in sperm from mice lacking the sperm-specific C{alpha}2 catalytic subunit of protein kinase A (PRKACA). Agonist responses also are strongly suppressed in wild-type sperm by the protein kinase inhibitor H-89. These results show that adenosine and catecholamine analogs activate sperm motility by mechanisms that require extracellular Ca2+, the atypical sperm adenylyl cyclase, cAMP, and protein kinase A.

ACIII, adenosine, C{alpha}2, catecholamines, cyclic adenosine monophosphate, gamete biology, sAC, signal transduction, soluble adenylyl cyclase, sperm motility and transport

INTRODUCTION

Although fertilization is essential for survival of species, the signaling pathways and molecular events underlying the meeting and union of the gametes remain poorly understood. After developing in the male reproductive tract, mammalian sperm are incapable of fertilizing an oocyte until they undergo additional obligatory changes, known as capacitation, that occur in the female reproductive tract [1]. An activation of rapid motility is one of the earliest steps of capacitation, occurring when ejaculated sperm are released into the reproductive fluids in vivo or incubated in bicarbonate-containing media in vitro. The low-amplitude flagellar beat becomes fast and highly symmetrical [2]. This activated flagellar waveform produces rapid swimming in straight trajectories and may aid the sperm in penetrating the mucus-filled cervix and in ascending the lower portions of the female genital tract [1, 3].

The present work concerns actions of two classes of small molecules, purine nucleosides and catecholamines, that may contribute to capacitation of sperm. Past work indicates that the nucleoside adenosine and catecholamines such as epinephrine (E) and norepinephrine (NE) are present in the reproductive fluids at the time of fertilization and could have important roles in sperm capacitation and fertilization. Adenosine was found at high micromolar concentrations in both male [4] and female reproductive fluids [5]. Catecholamines were reported in the female reproductive tracts of the rabbit [6], pig [7], cow [8, 9], and human [10]. The catecholamine content of the reproductive fluids reportedly changes during the ovulatory cycle [6, 8, 9]. Portions of both female and male reproductive tracts, especially the ovary, the isthmus of the oviduct, and the vas deferens, are heavily innervated by sympathetic adrenergic inputs that release primarily NE and the cotransmitter ATP, which can generate adenosine [10, 11]. Oviductal cells also release cAMP, which is metabolized to adenosine by ecto-phosphodiesterases and ecto-5'-nucleotidases [12].

Some previous work shows that adenosine and catecholamine agonists can stimulate sperm. For example, adenosine and analogs were reported to variously increase the percentage of motile cells, intracellular cAMP content, protein tyrosine phosphorylation, capacitation, or fertilizing ability of mouse [13–15], bull [16], and human sperm [17–20]. Actions of catecholamines on sperm capacitation have been less studied. However, norepinephrine and epinephrine were reported to induce capacitation in mouse [21], hamster [22–24], and bull sperm [25]. The catecholamines were also reported to increase spontaneous acrosome reactions [22, 24, 25] and in vitro fertilization rates [21, 26].

In somatic cells, various subtypes of adenosine and adrenergic receptors couple via G proteins to conventional transmembrane adenylyl cyclases (ADCY1–9) to increase or decrease cAMP content in response to agonists [27]. However, the predominant adenylyl cyclase enzyme of sperm, SACY, is an atypical member of the adenylyl cyclase family that lacks transmembrane domains and is unaffected by G proteins [28–30]. Instead, SACY is directly stimulated by bicarbonate [29, 30], one established physiological activator of sperm motility [2] and of other early events in the capacitation sequence [31]. Components of G protein-coupled olfactory signal transduction have also been reported in sperm [32–35], including identification of mRNA and detection of the olfactory ADCY3 protein in spermatogenic cells [36–38]. Thus, the question arises whether the adenosine and catecholamine agonists act through SACY or through a G protein-coupled receptor linked to stimulation of a conventional adenylyl clyclase, such as ADCY3. The aims of the current study were to investigate the actions of adenosine and catecholamine agonists on the activation of mouse sperm motility examined in vitro and to dissect the underlying signaling pathways using biophysical, pharmacological, and molecular approaches.

MATERIALS AND METHODS

Materials

H-89 and 2', 5'-dideoxyadenosine were from Calbiochem (San Diego, CA), and fura-2 acetoxymethyl (AM) ester and Pluronic F127 from Molecular Probes (Eugene, OR). The enzyme immunoassay, Correlate-EIA cAMP Kit, was from Assay Designs, Inc. (Ann Arbor, MI). All other chemicals were from Sigma (St. Louis, MO).

Animals and Sperm Preparation

Wild-type sperm were obtained from male Swiss-Webster retired-breeder mice, unless noted otherwise. Mutant sperm were obtained from 10 to 16 week old males from three lines of knockout mice bred on a C57BL/6 strain background. ADCY3 null animals were developed by Daniel Storm and coworkers at the University of Washington [39]. Male null mice were generated by mating ADCY3 heterozygous males and ADCY3 null females [38] and were compared with their wild-type C57BL/6 littermates. Mice null for SACY were engineered by Esposito et al. [40]. Mice null for C{alpha}2, the sperm-specific splice variant of the catalytic subunit of protein kinase A (PRKACA), and their heterozygous C57BL/6 littermates were produced by G. Stanley McKnight and coworkers [41]. All animal procedures were in accordance with accepted standards of humane animal care and were approved by the Animal Care and Use Committee at the University of Washington.

As in prior work [2, 42] sperm were obtained from the caudal epididymis and vas deferens. Briefly, after CO2 asphyxiation the tissues were excised, then cleaned and rinsed with medium Na7.4 (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgSO4, 20 Hepes, 5 glucose, 10 lactic acid, 1 pyruvic acid, adjusted to pH 7.4 with NaOH. Epididymal semen was allowed to exude for 15 min (at 37°C, 5% CO2) into a ‘swimout/capacitation' medium (Na7.4 with 5 mg/ml of BSA and 15 mM NaHCO3). Sperm were then sedimented, washed twice, dispersed, and examined at room temperature. Depolarization-evoked responses for Ca2+ photometry experiments were produced with medium K8.6 (in mM): 135 KCl, 5 NaCl, 2 CaCl2, 1 MgSO4, 30 TAPS [N-tris(hydroxymethyl)-methyl-3-aminopropane sulfonic acid], 10 glucose, 10 lactic acid, 1 pyruvic acid, adjusted to pH 8.6 with NaOH.

Measurements of cAMP in Sperm

For each experimental trial, sperm from 3 to 5 animals were prepared as described above, and pooled in Na7.4 medium. Fifty or 100 µl of sperm suspension was added to an equal volume of medium with or without the agonists: 25 µM 2-chloro-2'-deoxyadenosine (Cl-dAdo), 0.2 µM isoproterenol (ISO), or 15 mM NaHCO3. Treatments were run in duplicate or triplicate on samples of 1.3 to 3.3 x 106 sperm, incubated at room temperature for 0–30 min. The reaction was stopped and the cells lysed with 0.5 or 1 ml of ice-cold 1 M HCl:absolute ethanol (1:100 v/v%). The lysed cell suspensions were put on ice for >30 min, then centrifuged under vacuum at 37°C for >5 h to dryness. Samples were reconstituted in 250 µl of sodium acetate buffer, transfered to microtiter plates, and cAMP was measured using the enzyme immunoassay kit according to the manufacturer's instructions, with the following minor changes. All samples and cAMP standards contained medium of the same ionic composition to control for alterations in cAMP-antibody binding and sensitivity. Medium Na7.4 containing 15 mM NaHCO3 was similarly desiccated to dryness under vacuum and reconstituted with the sodium acetate buffer to run the kit cAMP standards. All results comprise at least three to six experimental trials.

Ester Loading of cAMP

As in prior work [42] sperm were incubated in 60 µM cAMP-acetoxymethyl ester (cAMP-AM) for loading. After 30 min, an aliquot of the cell suspension was added to the sample chamber containing medium Na7.4. Cells were imaged within ~5 min to minimize the reduction of intracellular cAMP content following the dilution of external cAMP-AM.

Waveform Analysis

The flagellar waveform was analyzed as described previously [2, 43]. Briefly, stop-motion images of single, loosely-tethered sperm were collected with an inverted microscope, a pulsed LED source, and cooled CCD camera. A semi-automated analysis program written in Igor (Wavemetrics; Lake Oswego, OR) allowed tracing of the flagellar images, determined various waveform parameters, and applied automated fitting of a sine function to the beat cycle to calculate flagellar beat frequency.

Dye Loading and Calcium Photometry

Similar to prior work [42] fura-2 AM was dispensed from 2 mM stocks in DMSO, dispersed in 10–15% Pluronic F127, then diluted to 10 µM in 0.5 ml of the sperm suspension in medium Na7.4. Cells were loaded with the dye at room temperature in the dark for 45 min, then sedimented. After resuspension in 0.3 ml of fresh medium, incubation continued for another 45 min before use. Five microliters of cell suspension was added to an uncoated glass coverslip inside a glass-bottomed incubation chamber containing ~3 ml of Na7.4. After ~3 min, test solutions were applied with a multi-barreled local perfusion device. Excitation light of 340 and 380 nm was delivered from a computer-controlled monochromator (T.I.L.L.; Gräfelfing, Germany), and >450 nm emitted light was collected by a photodiode detector from an adjustable viewfinder that selected a rectangular region containing a cluster (4–15) of loosely-tethered sperm. The raw photometric signals were corrected for cell-free background. The calibrated signal reports the spatially-averaged internal free Ca2+ concentration. Further analyses were performed in Igor (Wavemetrics; Lake Oswego, OR).

Statistical Analysis

Statistical analyses were performed in Excel (Microsoft; Redmond, WA). Beat frequencies and cAMP contents are presented as mean ± SEM. Intracellular free Ca2+ concentrations are presented as the means of multiple experiments. The data from each experiment were pooled and analyzed by ANOVA followed by a Student t-test, for paired comparisons. A P value of < 0.05 was considered statistically significant.

RESULTS

Increase of Flagellar Beat Frequency and cAMP Content of Mouse Sperm

We begin by examining the actions of the adenosine analog Cl-dAdo and the ß-adrenergic receptor agonist, ISO, on the flagellar beat of epididymal sperm. Beat frequencies were obtained from analysis of video movies of mouse sperm loosely tethered to a glass surface. Figure 1 shows single video frames taken from movies of representative individual sperm. Each movie (viewable in supplemental files at http://www.biolreprod.org.) contains one segment captured during perfusion with medium Na7.4 alone, and a second segment captured at 60 sec of perfusion with Na7.4 containing 25 µM Cl-dAdo (Fig. 1A) or 0.2 µM ISO (Fig. 1B).


Figure 1
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  		FIG. 1. Representative stop-motion video images of loosely tethered wild-type mouse sperm that received stimulus with 25 µM Cl-dAdo (A) or 0.2 µM ISO (B). Video movies from which these images were taken can be viewed in supplemental files at http://www.biolreprod.org. Frame sizes are 48 x 48 µm

Figure 2A shows the time courses of the actions of various concentrations of Cl-dAdo on the speed of the flagellar beat. When applied at 25 µM, Cl-dAdo robustly increased beat frequency from 2.7 ± 0.1 to 9.3 ± 0.5 Hz. The action was rapid (t1/2 ~20 sec) and dose-dependent. The half maximal excitatory concentration (EC50) was ~10 µM. Similarly, 0.2 µM ISO (with 1 mM ascorbic acid as an antioxidant) rapidly and reversibly increased beat frequency from 3.1 ± 0.1 to 9.6 ± 0.8 Hz (Fig. 2B). Treatment with 1 mM ascorbic acid alone was only marginally effective (2.6 ± 0.1 before vs. 3.5 ± 0.2 Hz after 60 sec of treatment). As for Cl-dAdo, the action of ISO was dose-dependent with an EC50 of ~0.05 µM. The actions of both agonists were slowly reversible. The flagellar beat returned toward the prestimulus resting rate within ~5 min after removal of the agonist (data not shown).


Figure 2
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  		FIG. 2. 2-Chloro-2'-deoxyadenosine and isoproterenol increase the beat frequency and accumulation of cAMP in mouse sperm. Averaged beat frequencies (mean ± SEM) of single randomly-selected sperm, each examined before and during application of various concentrations of: Cl-dAdo (A) (n = 3–4 cells in three independent experiments) or ISO (B) with 1 mM ascorbic acid (n = 6–14 cells in 2–7 independent experiments). C) Averaged cAMP content of sperm treated for various times with 25 µM Cl-dAdo (triangles), 0.2 µM ISO (squares), or 15 mM NaHCO3 (circles) (n = 5–14, 9–24 animals)

Past work shows that the acceleration of the flagellar beat by bicarbonate is mediated by increases in cAMP content [41]. Therefore we asked if adenosine and catecholamine agonists also increase sperm cAMP. Figure 2C compares the time courses of cAMP accumulation in sperm treated with Cl-dAdo, ISO, or NaHCO3. Intracellular cAMP increased within 45–60 sec of treatment with each of the three agonists. A 45-sec treatment with 25 µM Cl-dAdo or 0.2 µM ISO (with 1 mM ascorbic acid) significantly increased sperm cAMP content from 2.5 ± 0.1 to 3.9 ± 0.2 or 3.7 ± 0.3 pmol/107 cells, respectively. A 45-sec treatment with 1 mM ascorbic acid alone was ineffective (2.7 ± 0.1 pmol/107 cells; P > 0.05). As positive controls, 30-sec and 60-sec treatments with 15 mM NaHCO3 raised cAMP content from 2.5 ± 0.1 to 4.2 ± 0.2 and 4.5 ± 0.4 pmol/107 cells. During continued exposure to Cl-dAdo, ISO or NaHCO3, cAMP rose to a peak, fell, and then rose again at 15–30 min (data not shown).

Transmembrane Adenylyl Cyclase

The rise of cAMP produced by treatments with Cl-dAdo and ISO requires activity of an adenylyl cyclase. This could be SACY or a conventional adenylyl cyclase, such as ADCY3. To test if ADCY3 is required, we compared the beat frequency of wild-type and ADCY3 null sperm before and during treatment with 25 µM Cl-dAdo or 0.1 µM ISO. The agonist responses were hardly affected by the absence of ADCY3 (Fig. 3, A and B). The time courses of the response were similar for wild-type and ADCY3 null sperm. The elevated beat frequency at 60 sec of treatment was only marginally different for wild-type and knockout sperm (8.3 ± 0.4 Hz vs. 7.2 ± 0.4 Hz for Cl-dAdo, and 7.1 ± 0.5 Hz vs. 8.6 ± 0.8 Hz for ISO; not statistically significant, P > 0.05).


Figure 3
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  		FIG. 3. Transmembrane adenylyl cyclase is not required for agonist-mediated acceleration of the flagellar beat. Parallel experiments examined beat frequency of wild-type (open circles) and ADCY3 null (closed circles) sperm treated with control Na7.4 medium supplemented at the arrow with: 25 µM Cl-dAdo (A) or 0.1 µM ISO (B) (n = 4–12 cells in two independent experiments). C) Beat frequency of wild-type sperm treated with Na7.4 supplemented where indicated with 100 µM SQ-22536, and with 25 µM Cl-dAdo (triangles), 0.2 µM ISO (squares), or 15 mM NaHCO3 (circles) (n = 4–8 cells in two independent experiments). D) Beat frequency of wild-type sperm treated with Na7.4 supplemented where indicated with 50 µM 2',5'-dideoxyadenosine (ddA), and with 25 µM Cl-dAdo (triangles), 0.2 µM ISO (squares), or 15 mM NaHCO3 (circles) (n = 7–12 cells in 2–3 independent experiments)

As a second test for the involvement of a conventional adenylyl cyclase, we used a pharmacological approach. The adenylyl cyclase inhibitors SQ-22536 (9-tetrahydro-2'-furyl adenine) and 2',5'-dideoxyadenosine are directed to the P-site of conventional transmembrane cyclases. Single cells were treated sequentially with the inhibitors alone and with either 25 µM Cl-dAdo, 0.2 µM ISO, or 15 mM HCO3–. Figures 3, C and D show that the agonist-mediated increases in beat frequency were unaffected by the application of 100 µM SQ-22536 or 50 µM 2',5'-dideoxyadenosine. As for sperm not treated with the inhibitors, all three agonists were able to accelerate the mean beat frequency from ~4 to >8 Hz within 90 sec in the presence of SQ-22536, and from ~3 to >7 Hz in the presence of 2',5'-dideoxyadenosine. SQ-22536 alone slightly increased the flagellar beat from a mean of 2.9 ± 0.1 to 4.1 ± 0.2 Hz at 150 sec when the agonist application began. The response to 2',5'-dideoxyadenosine alone was also a variable slight increase in the beat rate. The inability of these inhibitors to block bicarbonate action is consistent with the interpretation that SACY, which lacks a P-site, is insensitive to them. Another cell-permeant, non-P-site inhibitor of conventional adenylyl cyclases, MDL-12330A [cis-N-(2-phenylcyclopentyl)-azacyclotridec-1-en-2-amine hydrochloride] also did not block agonist-evoked acceleration of flagellar beat. In the presence of 100 µM of this inhibitor, 25 µM Cl-dAdo increased the beat to 7.3 ± 0.6 Hz within 45 sec, and 0.1 µM ISO increased the beat to 6.3 ± 0.4 Hz. Unlike SQ-22536 and 2',5'-dideoxyadenosine, MDL-12330A alone did not increase the beat. Taken together, these experiments show no requirement for conventional adenylyl cyclases in the responses to adenosine and catecholamine agonists.

Sperm Adenylyl Cyclase

To test the alternate hypothesis that SACY is necessary for agonist responses, we compared sperm from wild-type and SACY null mice. Before exposure to the agonists, resting beat frequencies of wild-type and SACY null sperm were similar, 3.2 ± 0.1 and 2.7 ± 0.1 Hz, respectively (Fig. 4A). However, the agonists failed to accelerate the beat of SACY null sperm. The beat rates for wild-type and SACY null sperm were 7.6 ± 0.3 vs. 2.7 ± 0.1 Hz at 60 sec of treatment with Cl-dAdo (Fig. 4A) and 7.4 ± 0.4 vs. 2.8 ± 0.1 Hz with ISO (Fig. 4B). In contrast, the beat rate of the null sperm still increased when the cAMP content was elevated by incubation with the cell-permeant cAMP-AM ester (from 2.6 ± 0.1 Hz to 6.3 ± 0.4 Hz; Fig. 4C), indicating that the pathways downstream of cAMP production are intact in the SACY null sperm.


Figure 4
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  		FIG. 4. Flagellar actions of adenosine and catecholamine agonists require SACY. Parallel experiments monitored beat frequency of wild-type (open circles) and SACY null (closed diamonds) sperm treated with Na7.4 medium supplemented at the arrow with: 25 µM Cl-dAdo (A) (n = 17–19 cells in 3–5 independent experiments) or 0.1 µM ISO (B) (n = 16–23 cells in four independent experiments). C) Beat frequency of wild-type (left) and SACY null (right) sperm randomly sampled after 1–10 min incubation in Na7.4 alone (open bars), 30 min incubation with 60 µM cAMP-AM ester (striped), or 5–10 min incubation in medium containing 100 µM IBMX (solid) (n = 20–50 cells in 3–6 independent experiments)

To investigate whether adenylyl cyclases other than SACY contribute to the resting cAMP content and resting beat frequency, SACY null sperm were treated with the broad-spectrum phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Wild-type and SACY null sperm were sampled after 1–10 min incubation in Na7.4 alone or after 5–10 min incubation in Na7.4 containing 100 µM IBMX. The accelerating action of the PDE inhibitor on the flagellar beat of wild-type cells (to 6.8 ± 0.4 Hz) was completely absent from null sperm (2.8 ± 0.1 Hz; Fig. 4C). In experiments not shown, we found that forskolin (50 µM), an activator of transmembrane adenylyl cyclases, did not stimulate the flagellar beat of wild-type sperm (2.5 ± 0.1 vs. ~2.7 ± 0.1 Hz after 1–10 min of treatment). We conclude that adenylyl cyclases other than SACY are not involved in determining the resting flagellar beat frequency.

Extracellular Ca2+ as a Cofactor

The activation of sperm motility by HCO3– requires extracellular Ca2+ [43] and SACY is a Ca2+-sensitive enzyme [44]. Therefore, we examined whether the activation by Cl-dAdo and ISO also requires extracellular Ca2+. Figure 5A compares the mean beat frequency of sperm randomly sampled after 1–10 min incubation in Na7.4 alone or with 25 µM Cl-dAdo or 0.2 µM ISO. In the nominal absence of extracellular Ca2+, the agonists were unable to speed the flagellar beat. The beat frequencies with and without Ca2+ were 8.3 ± 0.6 vs. 2.6 ± 0.1 Hz with Cl-dAdo, and 7.0 ± 0.5 vs. 2.7 ± 0.1 Hz with ISO. Similar experiments examined single sperm treated sequentially with Na7.4 medium alone, then with 25 µM Cl-dAdo, first in the absence then in the presence of 2 mM added Ca2+ (Fig. 5B). Cl-dAdo was unable to increase beat frequency in the absence of extracellular Ca2+. When 2 mM Ca2+ was restored, the stimulatory action of Cl-dAdo returned.


Figure 5
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  		FIG. 5. Accelerating actions of adenosine and catecholamine require extracellular Ca2+. A) Beat frequency (mean ± SEM) of sperm randomly sampled after 1–10 min incubation in standard medium Na7.4 (left) or in a nominally Ca2+-free version of the medium Na7.4 (right). Further additions were: none (open bars), 25 µM Cl-dAdo (striped), or 0.2 µM ISO (closed) (n = 14–21 cells in three independent experiments). B) Averaged beat frequency of single sperm treated sequentially with Na7.4 alone, then with 25 µM Cl-dAdo in modified Na7.4 containing 0 and then 2 mM added Ca2+, as indicated (n = 4 cells in two independent experiments). The dashed lines enclose the period of treatment with nominally Ca2+-free medium

Adenosine and adrenergic agonists are known to modulate intracellular Ca2+ in a variety of cell types [27]. The Ca2+-dependence of the action of these agonists in sperm might indicate that a required increase in intracellular Ca2+ stimulates the Ca2+-sensitive SACY enzyme to increase cAMP. To test this hypothesis we used fura-2 ratiometric photometry to monitor sperm intracellular free [Ca2+] during stimulation with the agonists. Figure 6 shows the averaged responses from several clusters of cells locally perfused with medium Na7.4, then for 60 sec with Na7.4 that was supplemented with 25 µM Cl-dAdo or 0.2 µM ISO. Finally as a positive control, sperm received a 10-sec depolarizing stimulus with medium K8.6 to open voltage-gated channels that allow Ca2+ entry. The agonists did not increase sperm intracellular [Ca2+]. The upstream mechanism of action of adenosine and catecholamine agonists apparently does not involve an increase in intracellular Ca2+ or mobilization from intracellular stores.


Figure 6
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  		FIG. 6. Adenosine and catecholamine agonists do not increase intracellular Ca2+. Intracellular free Ca2+ concentration is reported by fura-2 ratiometric photometry. Averaged responses from clusters of cells locally perfused with Na7.4, or with Na7.4 supplemented with: 25 µM Cl-dAdo (A) or 0.2 µM ISO (B), except during a 10-sec depolarizing stimulus with medium K8.6, as indicated (n = 11–13 clusters of cells in two independent experiments). Rates of [Ca2+] rise (nM sec–1) are noted

Sperm-Specific C{alpha}2 PRKACA Subunit

To test the hypothesis that the signaling pathways for adenosine and catecholamine agonists require phosphorylation by PRKACA, the sperm from mice lacking C{alpha}2, the sperm-specific splice variant of PRKACA were compared with those of their heterozygous littermates. Sperm were treated sequentially with Na7.4 alone, then with 25 µM Cl-dAdo or 0.1 µM ISO (Fig. 7). As for the SACY null sperm, the C{alpha}2 null sperm did not accelerate in response to Cl-dAdo or ISO. Before exposure to the agonists, the mean resting beat frequency of C{alpha}2 null sperm was slightly higher than for sperm from their littermates, 3.4 ± 0.1 vs. 2.7 ± 0.2 Hz, respectively. However, the accelerating action of Cl-dAdo on control sperm (7.1 ± 0.2 Hz at 60 sec) was completely absent from the null sperm (3.5 ± 0.2 Hz; Fig. 7A). ISO was similarly effective in heterozygous sperm (6.4 ± 0.5 Hz at 60 sec), but ineffective in C{alpha}2 null sperm (3.4 ± 0.2 Hz; Fig. 7B).


Figure 7
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  		FIG. 7. Sperm-specific C{alpha}2 splice variant of PRKACA is required for adenosine and catecholamine actions. Parallel experiments monitored beat frequency of C{alpha}2 null (closed diamonds) or heterozygous (Het) littermate (open circles) sperm treated with Na7.4 medium supplemented at the arrow with: 25 µM Cl-dAdo (A) (n = 5–14 cells in 2–4 independent experiments) or 0.1 µM ISO (B) (n = 4–15 cells in 2–4 independent experiments)

PRKACA Inhibitor

Using a pharmacological approach as a second test for the involvement of PRKACA in the agonist-mediated pathways, sperm were treated with the PRKACA inhibitor H-89. Beat frequency was determined for sperm randomly sampled after 1–10 min incubation in Na7.4 alone or with 25 µM Cl-dAdo or 0.1 µM ISO, in the presence and absence of 30 µM H-89 (for the inhibitor-treated cells incubation with H-89 began 5 min before agonist exposure). Beat frequencies of control Na7.4- and H-89-treated cells were identical at 2.7 ± 0.1 Hz (P > 0.05). The agonists did not speed beat frequency in the presence of the PRKACA inhibitor, providing further evidence of a required role of PRKACA (Table 1).


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  		TABLE 1. PRKACA inhibitor blocks agonist action on sperm beat frequency.a

DISCUSSION

We have found that the adenosine analog, Cl-dAdo, and the catcholamine agonist, ISO, potently accelerate the sperm flagellar beat. Responses are maximal within 60 sec and accompanied by increases in cAMP content. Activation of the machinery of sperm motility by these agonists requires extracellular Ca2+, PRKACA, and SACY, rather than a conventional adenylyl cyclase.

Our results confirm and extend previously reported actions of adenosine analogs on sperm. Vijayaraghavan and Hoskins [16] found that 20–300 µM of adenosine analogs, including 2-chloroadenosine and 2'-deoxyadenosine, increased the percentage of motile cells and the cAMP content of bull sperm. Nucleosides other than adenosine were ineffective. Further studies in mouse [15, 45], bull [46], and human [17, 19, 20] sperm reported that adenosine analogs variously increased the percentage of motile sperm, the cAMP content, capacitation, or the phosphorylation of proteins such as glycogen synthase kinase-3{alpha}, with EC50 values ranging from 0.3–2500 µM. Although knockout mice carrying targeted disruptions of A1, A2A, or A3 adenosine receptors remain fertile [47], mice lacking the A1 receptor are reported to have decreased male fertility and to have deficits in sperm capacitation in vitro [48]. Thus adenosine may still have a nonessential role in sperm capacitation in vivo. Alternatively, adenosine may have roles that do not involve these receptors.

Some early studies suggested actions of catecholamines on the later stages of sperm capacitation. For instance, the subjective assessments of Cornett and Meizel [22] indicated that 0.5–50 µM E and 50 µM NE or ISO increased the number of hamster sperm with hyperactivated "whip-lash" motility. The agonists did not increase the percentage of motile cells, but did increase the proportion of spontaneous acrosome reactions (a likely indication of toxicity). At more physiologically-relevant agonist concentrations, 40–80 nM NE but not E increased lysophosphatidylcholine-induced acrosome reactions of bull sperm, but also increased the proportion of spontaneous acrosome reactions in cells capacitated for 2 h in heparin [25]. Other work reported that 1 µM E increased cAMP content, capacitation assessed by chlortetetracycline staining, and in vitro fertilization for mouse sperm [21]. No prior studies have reported rapid acceleration of the flagellar beat by catecholamines. The present work thus may be the first study of the time course of catecholamine and adenosine actions on flagellar function of mouse sperm.

No Involvement of Transmembrane Adenylyl Cyclase

Is there a role for transmembrane adenylyl cyclases in spermatogenic cells and sperm? Round and elongating spermatids possess mRNA for the olfactory ADCY3 and for the G{alpha}olf homologue of Gs [36–38]. Western blot analysis and immunocytochemistry indicated that post-meiotic germ cells also may produce the ADCY3 protein [49, 50]. However, a role for ADCY3 in mature sperm remains controversial. The recently engineered ADCY3 knockout mouse is anosmic. The males are subfertile and reportedly have a sperm motility deficit [38]. Nevertheless, we find that for sperm from the ADCY3 null mice the basal beat frequency is normal and is increased effectively by Cl-dAdo and ISO. The marginally reduced response to Cl-dAdo could have any of several explanations, including a developmental defect resulting from the absense of ADCY3 during spermatogenesis. Regardless of the explanation, these results indicate that ADCY3 has no required role in the flagellar responses to adenosine and catecholamine agonists.

We also find pharmacological evidence that ADCY3 and other transmembrane adenylyl cyclases do not participate in the responses to these agonists. The inhibitor SQ-22536 acts by binding to the P-site of ADCY3 and other transmembrane adenylyl cyclases, forming a dead-end complex with pyrophosphate and inhibiting conversion of ATP to cAMP [51]. We show that this inhibitor does not block the increase in beat frequency produced by Cl-dAdo and ISO. The slight stimulation of the beat of wild-type sperm by SQ-22536 may result from cross-activation of a hypothetical cell surface adenosine receptor [51], or may be due to its entry and inhibition of PDE. Two other transmembrane adenylyl cyclase inhibitors, the P-site binding, 2',5'-dideoxyadenosine, and the non-P-site inhibitor, MDL-12330A, were similarly ineffective at blocking adenosine and catecholamine agonist action. Evidently conventional adenylyl cyclases are not needed for agonist-mediated acceleration of the flagellar beat.

We find that forskolin, an activator of transmembrane adenylyl cyclases, was unable to increase the flagellar beat of wild-type sperm. The SACY null sperm also did not accelerate in response to the PDE inhibitor, IBMX. Thus, there is no evidence that adenylyl cyclases other than SACY contribute to resting cAMP levels and to the control of the resting flagellar beat. Measurements of resting cAMP content support this interpretation. The cAMP content of SACY null sperm is severely depressed (unpublished work of Xie F, Garcia MA, and Conti M). In contrast the cAMP content of ADCY3 null sperm is indistinguishable from that of wild-type sperm [38].

Requirement for SACY

The rapid activation of motility by bicarbonate [2] and several other bicarbonate-dependent components of capacitation [31] apparently involve a direct activation of SACY by the bicarbonate anion [29–31, 40]. No agonists other than bicarbonate or bicarbonate-like anions are known to stimulate the activity of native or recombinant SACY. Nonetheless, we now show that catecholamine and adenosine agonists increase cAMP content and accelerate wild-type sperm, but fail to accelerate SACY null sperm, indicating that the flagellar actions of Cl-dAdo and ISO require SACY. The SACY null sperm accelerated when cAMP was generated from its cell permeant AM-ester, indicating that the flagellar machinery downstream of cAMP was intact. Rescue of the motility of SACY null sperm by cAMP derivatives has been noted in prior work [40, 52].

Stimulation of SACY Activity

The mechanisms that couple the adenosine and catecholamine agonists to SACY are not known. It is possible that unique receptors for adenosine and catecholamines link to the SACY enzyme to stimulate its activity. Alternatively, these agonists might have an intracellular site of action. Possible scenarios include direct or indirect stimulation of the SACY enzyme. Alternatively these agonists might act by inhibition of PDE to increase accumulation of the cAMP that is produced by basal SACY activity.

When applied at relatively high concentrations (7–100 µM) catecholamine agonists inhibited PDE activity in other cell types [53, 54], and >600 µM of adenosine analogs were found to inhibit sperm PDE activity [16]. We now find that metabolically stable catecholamine and adenosine agonists accelerate the flagellar beat at significantly lower concentrations than the half maximal inhibitory concentrations (IC50) reported for PDE inhibition. Thus, it seems unlikely that the agonists are acting solely as PDE inhibitors. However, the possible involvement of PDEs in the action of these agents requires further investigation.

Some past work examined direct actions of various agonists on sperm adenylyl cyclase. However, adenylyl cyclase activity assays indicated that high concentrations (~0.2–1 mM) of adenosine, and catecholamines such as isoproterenol are inhibitory, rather than stimulatory [55, 56]. Alternatively, indirect action of catecholamine and adenosine agonists might involve the established stimulatory action of Ca2+ on SACY [44]. However we find that spatially averaged intracellular [Ca2+] does not rise during application of these agonists.

Despite the lack of a detectable rise in internal [Ca2+], we find that extracellular Ca2+ is required for the accelerating actions of adenosine or catecholamine analog on the flagellar beat. External Ca2+ is also required for the accelerating action of bicarbonate on the flagellar beat of mouse sperm [43], and for bicarbonate to increase cAMP content in sperm of the guinea pig [57] and mouse [42]. These results indicate that agonist action in sperm is a Ca2+-dependent process. The results also are consistent with the idea that the atypical Ca2+-sensitive SACY enzyme is required for agonist action. Although no increases in spatially-averaged Ca2+ signals were observed during application of agonists, we cannot exclude the possibility that the agonists produce local increases in [Ca2+] that enhance SACY activity. In concept, Ca2+ might act instead soley at an extracellular site. The mechanism of the Ca2+-dependent and -independent events upstream of the sperm cyclase remains to be elucidated.

Requirement for Sperm-Specific Splice Variant of PRKACA

Much past work indicates that PRKACA and PRKACA-mediated protein phosphorylations are crucial molecular components underlying the regulation of sperm motility and capacitation [31, 58, 59]. Most compellingly, this lab showed that cAMP-mediated actions of bicarbonate require the sperm-specific C{alpha}2 splice variant of the PRKACA catalytic subunit [2, 41]. In the present study we find that Cl-dAdo and ISO were ineffective at accelerating the beat of C{alpha}2 null sperm. Pharmacological blockade with the PRKACA inhibitor H-89 in wild-type sperm provides additional evidence for the involvement of PRKACA-mediated phosphorylation in agonist action. Thus we conclude that adenosine and catecholamine agonists also act in a signaling pathway that requires downstream engagement of PRKACA. This is the first demonstration of a required role of PRKACA in the action of adenosine and catecholamine agonists in stimulating sperm flagellar function.

Our results with adenosine and catecholamine agonists show much similarity to the known actions of bicarbonate. Each agonist increases the flagellar beat rate [2] and cAMP content [41, 42] within seconds. The flagellar actions of each also require extracellular Ca2+ [43] and the catalytic activities of SACY [40, 52] and PRKACA [2, 41]. We conclude that subsequent to the elevation of cAMP, adenosine and catecholamines share a common downstream pathway with bicarbonate. As expected for a shared mediating action of cAMP, the cell-permeant cAMP-AM ester mimics the action of the three agonists on motility.

In summary, adenosine and catecholamine agonists are candidate modulators of sperm motility and capacitation that may coordinate the meeting of sperm and egg. The signaling pathways for these agonists in somatic cells, involving G protein-coupled receptors linking to transmembrane adenylyl cyclases, apparently do not apply to sperm. For sperm, future work is needed to elucidate the upstream mechanisms that couple these agonists to SACY. Further investigation is also needed to determine whether these effects on sperm actually occur in the female tract from endogenous catecholamines and nucleosides. Determination of the molecular events and signaling pathways by which relatively immotile quiescent sperm become capable of fertilization, may contribute to our understanding of the complex process of sperm capacitation. Such understanding may aid in the development of improved contraceptives and treatments of motility-related clinical infertility.

ACKNOWLEDGMENTS

We thank Dr. Michael A. Nolan for generous help with phenotypic analysis of the sperm of the PRKACA C{alpha}2 null and heterozygous littermate mice.

FOOTNOTES

2 Correspondence: Donner F. Babcock, Department of Physiology and Biophysics, MS 357290, University of Washington, Seattle, WA 98195-7290. FAX: 206 685 0619; donner{at}u.washington.edu Back

1 Supported by U54-HD12629 of the Specialized Cooperative Centers Program in Reproduction Research of NICHD (B.H. and D.F.B.), and by NICHD grant HD31544 (M.C.). S.M.S. was supported in part by NIH 5T32 HD07183. A.E.C. was supported in part by NRSA T32 GM07270 from NIGMS. Back

Received: 23 September 2005.

First decision: 18 October 2005.

Accepted: 15 November 2005.

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