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Department of Physiology and Biophysics, University of Washington, Seattle, Washington 98195
ABSTRACT
Activation of rapid motility apparently is one of the first steps of sperm capacitation and can be studied in vitro. Previously we found that 2-chloro-2'-deoxyadenosine or the catecholamine isoproterenol activates mouse sperm motility in vitro via a pathway mediated by cAMP that requires extracellular Ca2+, the atypical sperm adenylyl cyclase, and sperm-specific protein kinase A. We now show that several other adenosine analogs and catecholamines accelerate the flagellar beat of mouse and human sperm. Unexpectedly, the potent adenosine receptor agonist CGS21680 does not accelerate the beat, and the adenosine receptor antagonist DPCPX does not diminish the accelerating action of 2-chloro-2'-deoxyadenosine. The pharmacological profile for activation by catecholamines is also unusual. Both agonists and antagonists of beta-adrenergic receptors elevate the beat frequency. Moreover, both l-(–) and d-(+) isomers of epinephrine, norepinephrine, and isoproterenol produce similar acceleration of the beat. In contrast, inhibitors of equilibrative nucleoside transporters effectively slow the onset of the accelerating action of adenosine analogs. Replacement of external Na+ with Li+ also diminishes the accumulation of cAMP and slows the resultant accelerating action of 2-chloro-2'-deoxyadenosine, suggesting the involvement of a Na+-dependent concentrative nucleoside transporter. Our results show that adenosine and catecholamine agonists act in a novel signaling pathway that does not involve G protein-coupled cell-surface receptors that link to conventional adenylyl cyclases. Instead, adenosine and analogs may be transported into sperm via equilibrative and concentrative nucleoside transporters to act on unknown intracellular targets.
adenosine, cAMP, catecholamines, CNT, ENT, flagellar beat, nucleoside transporter, sAC, SLC28A, SLC29A, soluble adenylyl cyclase, sperm capacitation, sperm motility and transport
When mammalian sperm are deposited into the female reproductive tract, they are incapable of fertilizing an oocyte until they undergo necessary physiologic and biochemical changes termed capacitation [1]. In the comprehensive sense as originally defined, capacitation includes those changes that occur between mating and fertilization that select and prepare the fertilizing sperm [2]. From this perspective, the activation of rapid motility is one of the first steps of capacitation, occurring when ejaculated sperm are released into the reproductive fluids. The fast symmetrical waveform of activated sperm [3] produces rapid swimming in straight trajectories and may aid in the penetration of the cervix, ascent into the upper portions of the female genital tract, and entry into the oviducts [1, 4, 5].
Small molecules present in the reproductive fluids may signal these changes in sperm motility and other events of capacitation [1]. Our past work indicates that bicarbonate is a probable physiologic activator of sperm motility [3] and suggests that adenosine and catecholamines may use the same cAMP-mediated pathway for activation of sperm [6]. Like bicarbonate [7], adenosine [8, 9] and catecholamines [10–14] are present at relatively high levels in the reproductive fluids of various mammals, including humans. In somatic cells, adenosine and catecholamines act at several subtypes of G protein-coupled receptors that couple to the conventional transmembrane adenylyl cyclases ADCY1–9 (formerly known as AC1–9) to regulate cAMP synthesis [15]. However, we have found that in sperm these agonists do not use a conventional transmembrane adenylyl cyclase enzyme but instead require the sperm or "soluble" adenylyl cyclase, SACY (formerly known as sAC) [6]. The SACY enzyme lacks transmembrane domains and is unaffected by G proteins [16, 17]. The mechanisms that couple the adenosine and catecholamine agonists to the stimulation of SACY are not known. One possibility is that the agonists act at conventional receptors on the plasma membrane that somehow link to the SACY enzyme. Some of these receptors are reportedly found in sperm [18–22], but their role in adenosine and catecholamine signaling in sperm has not been conclusively demonstrated.
Because the SACY enzyme is not stimulated by G proteins, it is possible that rather than acting at conventional receptors, adenosine and catecholamine analogs are entering sperm via transporters and acting at intracellular targets. Many somatic cells synthesize purine and pyrimidine nucleosides. Cells that lack nucleoside biosynthesis accumulate nucleosides using equilibrative nucleoside transporters (known as solute carrier family 29, SLC29A; formerly known as ENT), concentrative nucleoside transporters (SLC28A; formerly known as CNT), or both [23].
Our past work found that 2-chloro-2'-deoxyadenosine (Cl-dAdo) and isoproterenol [24] act through a cAMP-mediated signaling pathway in mouse sperm that requires extracellular Ca2+, the sperm adenylyl cyclase, and protein kinase A [6]. Using biophysical and pharmacological approaches, we now show that several additional adenosine and catecholamine agonists effectively accelerate the beat of sperm from both mouse and human and that this action likely involves transporters rather than conventional receptors on the plasma membrane.
CGS21680 was from Research Biochemicals Inc. (Natick, MA), and (-)-epinephrine bitartrate, (+)-epinephrine bitartrate, and (-)-norepinephrine bitartrate were from Calbiochem (La Jolla, CA). All other chemicals were from Sigma (St. Louis, MO). Dipyridamole, CGS21680 (2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine hydrochloride), and NBTI (4-nitrobenzyl-6-thioinosine) were prepared from stock solutions stored at –20°C in dimethyl sulfoxide (DMSO) at concentrations 
1000-fold those of the final concentrations used. The concentration of DMSO in experimental solutions was less than 0.1%. All other experimental solutions were made fresh on the day of the experiment.
Sperm were obtained from male Swiss-Webster retired-breeder mice as in prior work [3, 6]. All protocols 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. Briefly, after CO2 asphyxiation the caudal epididymis and vas deferens were excised, then cleaned and rinsed with medium Na7.4 (also known as medium HS): 135 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 20 mM HEPES, 5 mM glucose, 10 mM lactic acide, 1 mM pyruvic acid, and 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 BSA and 15 mM NaHCO3). Sperm were then sedimented, washed twice in Na7.4, dispersed, and examined at room temperature. For nominally sodium-free experiments, the following alterations were made to medium Na7.4: LiCl replaced the NaCl, Li-lactate replaced the Na-lactate, and the solution was adjusted to pH 7.4 with tetraethylammonium hydroxide and fortified with either 15 mM tetraethylammonium chloride or 15 mM tetraethylammonium bicarbonate.
Human semen samples were collected from six healthy men, 18–55 years old, and were generously provided by Dr. John Amory and coworkers (Department of Medicine, University of Washington). All procedures involving human subjects were approved by the Institutional Review Board at the University of Washington. Written informed consent was obtained before screening. The donors had general good health; normal medical history; no routine use of medication; normal physical examination, including testicular volume and prostate size; normal serum laboratory tests, including complete blood count, liver function tests, gonadotropins, total testosterone, luteinizing hormone, and FSH concentrations; and prostate-specific antigen <4.0 ng/ml. Subjects had normal seminal fluid analyses as defined by World Health Organization criteria (sperm count > 20 million/ml, motility > 50%, morphology > 40% normal forms) [25]. Exclusion criteria included history of alcohol or anabolic steroid abuse, prior history of infertility, or vasectomy. After at least 48 h of ejaculatory abstinence, the freshly collected semen sample was allowed to liquefy for 30–60 min at 37°C (5% CO2). The sample was divided into 0.25-ml aliquots, diluted 4-fold with medium Na7.4, and sedimented at 333 x g for 7 min. Cell pellets were resuspended in 0.75 ml of medium Na7.4, sedimented for 3 min, then dispersed in 0.5 ml of medium Na7.4 at a final concentration of 1.1 x 107 cells/ml or greater, and examined at room temperature.
The flagellar waveform was analyzed as described previously [3, 6]. Briefly, stop-motion images of single, randomly selected, loosely tethered sperm were collected with an inverted microscope, a pulsed LED source, and a cooled CCD camera (CoolSnap HQ; Photometrics, Tuscon, AZ). The images were initially processed to enhance intensity and contrast and to eliminate immotile background objects using either MetaMorph (Molecular Devices, West Chester, PA) or ImageJ (http://rsb.info.nih.gov/ij/). Then, a semiautomated analysis program written in Igor (Wavemetrics, Lake Oswego, OR) determined various waveform parameters, including flagellar beat frequency, the primary determinant of swimming speed. For imaging of mouse sperm, the sperm suspension was pipetted directly onto an uncoated 
5-mm round glass chip (Bellco, Vineland, NJ) inside a 35-mm culture dish with a no. 0 coverslip bottom. For human sperm, the sperm suspension was pipetted directly onto the surface of a 35-mm polystyrene cell culture dish (Corning Inc., Corning, NY) to allow loose attachment of sperm heads and optimal analysis of flagellar waveform.
Measurements of cAMP in Mouse Sperm
For each experimental trial, pooled sperm from four mice were prepared as described above. Five minutes before exposure to stimulus, the pooled sperm were divided for final sedimentation and resuspension in either medium Na7.4 or its modified, Li+-substituted version. Total exposure thus was limited to <6 min to minimize any consequences of long-term Li+ exposure. A total of 50 or 100 µl resuspended sperm (1.2 to 1.4 x 106 cells) was added to an equal volume of medium with or without the agonists 25 µM Cl-dAdo or 15 mM bicarbonate. After 60 sec at room temperature, cells were lysed by the addition of 5 vol of ice-cold 0.2 M HCl in 100% ethanol. After 30 min on ice, samples were lyophilized and analyzed by enzyme immunoassay (Correlate-EIA cAMP Kit; Assay Designs, Ann Arbor, MI) per the manufacturer's instructions for acetylated samples, with the following minor changes. All samples and cAMP standards contained medium of the same osmolarity and substrate composition to control for alterations in cAMP-antibody binding and sensitivity. Medium Na7.4 was desiccated to dryness under vacuum and reconstituted with HCl to run the kit cAMP standards. All results comprise two experimental trials from a total of eight mice.
Statistical analyses were performed in Excel (Microsoft, Redmond, WA). Beat frequencies are presented as mean ± SEM. The sample sizes report the total number of sperm analyzed for each treatment group. In the experiments examining human sperm treated with adenosine analogs and mouse sperm in Na+-free medium, the time taken to reach twice the resting beat frequency (doubling time) was calculated by normalizing the data points to the average resting beat frequency and then performing a linear regression analysis from 0–90 sec. For some experiments, the beat frequency did not actually double, and for these it is an extrapolated doubling time. For all experiments, the data from two or more independent experiments 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.
Adenosine and Catecholamine Agonists Accelerate the Beat of Mouse Sperm
We first examined the actions of Cl-dAdo and the catecholamine norepinephrine (NE) on the flagellar beat of mouse sperm. Figure 1A shows single video frames taken from movies (see supplemental Movies 1A and 1B) of representative individual sperm treated with these agonists. Each movie contains one segment captured during perfusion with control Na7.4 alone, and a second segment captured after 90 sec of perfusion with Na7.4 containing 10 µM Cl-dAdo or 1 µM NE. Visual examination indicates that these agonists robustly accelerated the beat of mouse sperm.
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Figure 1B shows the time courses of the actions of Cl-dAdo and NE on the flagellar beat. When applied at 25 µM, Cl-dAdo increased beat frequency from 2.7 ± 0.1 Hz to 10.0 ± 0.7 Hz (mean ± SEM). This action was rapid (with a t1/2 of 20 sec) and reversible. The beat frequency returned toward the prestimulus rate within 3 min after removal of Cl-dAdo (Fig. 1B). Similarly, NE at 1 µM (with 1 mM ascorbic acid as an antioxidant) rapidly and reversibly accelerated beat frequency from 2.9 ± 0.1 to 6.8 ± 0.6 Hz. The time course of this action and its reversal were similar to those for Cl-dAdo.
Several other adenosine analogs and catecholamines also accelerated the beat. Table 1 shows the beat frequency of single sperm examined before and then at the end of a 90-sec local perfusion with adenosine (Ado) or several analogs. Adenosine at 2.5 mM was the least effective, accelerating the beat from 2.3 ± 0.1 Hz to 4.1 ± 0.5 Hz, and Cl-dAdo at 25 µM was the most effective, accelerating the beat from 2.6 ± 0.1 Hz to 8.8 ± 0.3 Hz. The analogs N-ethylcarboxamidoadenosine (NECA), 2'-deoxyadenosine (dAdo), and 2-chloroadenosine (Cl-Ado) were intermediate in their ability to speed the beat. Similarly, 1–10 min of incubation with the catecholamines epinephrine (E), NE, or isoproterenol (ISO) substantially accelerated the flagellar beat (n = 21 to 53 cells in two to six independent experiments). The general adrenergic receptor agonist E (1 µM) was the least effective (5.0 ± 0.4 Hz vs. 2.8 ± 0.1 Hz in E vs. untreated sperm, respectively), and the β-selective adrenergic receptor agonist ISO (0.1 µM) was the most effective and potent (6.8 ± 0.3 Hz vs. 2.8 ± 0.1 Hz in ISO vs. untreated sperm, respectively). Norepinephrine (1 µM) was intermediate in its ability to speed the beat (6.2 ± 0.4 Hz).
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Adenosine and Catecholamine Agonists Accelerate the Beat of Human Sperm
We wanted to compare the responses of human and mouse sperm to adenosine and catecholamine agonists. Figure 2A shows single video frames taken from movies (see supplemental Movies 2A and 2B) of representative individual human sperm treated with medium Na7.4 alone or with 25 µM Cl-dAdo. For human sperm in medium Na7.4, the mean resting beat frequency (6.6 ± 0.4 Hz; Fig. 2B) was significantly greater (P < 0.0001) than for mouse sperm (2.8 ± 0.2 Hz; Table 1; see supplemental Movie 2C). Similar to their action on mouse sperm, the adenosine analogs Cl-dAdo and dAdo, the catecholamine ISO, and the anion bicarbonate each accelerated the beat of human sperm nearly 2-fold.
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Conventional Adenosine Receptors?
We turned to a pharmacological approach to test for conventional plasma membrane receptors in the adenosine-evoked speeding of the flagellar beat. Single cells were treated sequentially with the potent adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) alone and with the antagonist combined with 10 µM Cl-dAdo (Fig. 3A). The Cl-dAdo-mediated acceleration of the beat was not reduced by the application of DPCPX (2, 20, and 40 µM), with beat frequency increasing from 2.5 to 8.0 Hz within 60 sec in the presence of DPCPX (Fig. 3A). In addition, the adenosine receptor agonist CGS21680 at 10 µM was ineffective at accelerating the beat (2.7 ± 0.1 Hz after 90 sec of CGS treatment vs. 7.7 ± 0.6 Hz after 90 sec of Cl-dAdo; Fig. 3B). These observations are not consistent with the operation of a conventional adenosine receptor in the activation of motility by adenosine analogs.
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Conventional Adrenergic Receptors?
To determine whether conventional adrenergic receptors participate in the catcholamine-evoked increase in beat frequency, we tested selective 
- and β-adrenergic receptor agonists and antagonists. Figure 4A compares the effects of the β-adrenergic receptor agonist ISO at 0.1 µM and the -adrenergic receptor agonist phenylephrine (PE) at 10 µM. ISO effectively accelerated the beat (7.8 ± 0.6 Hz at 90 sec), but phenylephrine was ineffective (3.5 ± 0.2 Hz at 90 sec). Despite the apparent preference for β-agonists, several β-adrenergic receptor antagonists did not block the actions of catecholamines; indeed, several even accelerated the flagellar beat (Fig. 4, B and C). Within 90 sec, the beat rate increased from 3.3 ± 0.1 Hz to 9.2 ± 0.6 Hz with 1 µM propranolol, from 3.3 ± 0.2 Hz to 8.1 ± 0.6 Hz with metoprolol, and from 3.1 ± 0.1 Hz to 6.1 ± 0.5 Hz with atenolol. Butoxamine, another β-antagonist, also accelerated the flagellar beat (data not shown). The -adrenergic receptor antagonist phentolamine (25 µM) was only marginally effective (2.9 ± 0.1 Hz before to 4.4 ± 0.4 Hz after 90 sec of treatment; Fig. 4C).
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As another test for the involvement of adrenergic receptors, sperm were treated with l-(–) and d-(+) catecholamine isomers (Fig. 4D). At 0.2 µM, both the l- and d-isomers of ISO, NE, and E increased the beat frequency to a similar extent and with similar time courses (Fig. 4D). This lack of catecholamine isomer specificity also is inconsistent with involvement of a conventional adrenergic receptor in the activation of motility by catecholamines.
Equilibrative Nucleoside Transporter?
We reasoned that if adenosine does not act via a conventional membrane receptor, then it may need to enter the cell. This hypothetical requirement for entry into sperm suggests that inhibitors of equilibrative nucleoside transporters should slow adenosine action. Figure 5 shows the actions of the SLC29A inhibitors, dipyridamole and NBTI, on the adenosine analog-evoked acceleration of beat. Cells treated with 10 µM dipyridamole took more than three times as long to reach near-maximal beat rates with 25 µM Cl-dAdo (Fig. 5A). In contrast, dipyridamole did not affect the accelerating action of 3 mM bicarbonate (Fig. 5B). In similar experiments, 10 µM NBTI slowed the response to 500 µM dAdo, with sperm taking more than 240 sec to reach near-maximal beat rates compared with 120 sec with dAdo alone (Fig. 5C). The slowing of the onset of activation with these inhibitors is consistent with their ability to slow the entry of adenosine and analogs into the cell. NBTI also did not affect the action of bicarbonate on the beat (Fig. 5D), indicating that the response was specific and the inhibitors do not depress the flagellar machinery or prevent the activation of rapid motility in general. Thus, an equilibrative nucleoside transporter(s) may have a role in the upstream part of adenosine action in sperm.
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Concentrative Nucleoside Transporter?
Heterologous expression studies using human and rodent orthologs of concentrative nucleoside transporters found large differences in selectivity for adenosine analogs [26, 27]. Member 2 of the rodent concentrative nucleoside transporter family, SLC28A2 transports Cl-dAdo better than the human SLC28A2 ortholog. Therefore, if adenosine analogs use SLC28A2 to enter sperm, then we should find a corresponding faster action of Cl-dAdo in the acceleration of mouse sperm than of human sperm. The beat rate of mouse sperm increased from 2.8 ± 0.2 Hz before to 7.3 ± 0.6 Hz at 90 sec of Cl-dAdo treatment (Fig. 6A). The beat rate of human sperm increased from 5.8 ± 0.4 Hz before to 10.3 ± 1.2 Hz with the same treatment (Fig. 6A). The extrapolated time for reaching twice the resting beat frequency (t2X) with Cl-dAdo was 39 sec in mouse sperm and 140 sec in human sperm. The slower time course of Cl-dAdo action for human sperm compared with mouse sperm is in line with Cl-dAdo being transported more slowly by the human ortholog of SLC28A2 than by the rodent ortholog [27]. Using the naturally occurring adenosine analog dAdo in a similar series of experiments, we found that the rates of the increase in beat were similar in mouse and human sperm, with t2X values of 111 sec and 140 sec, respectively (P > 0.05; Fig. 6B). These similar kinetics of dAdo action on human and mouse sperm parallel the results of similar dAdo transport rates in human and rodent SLC28A2 [27] and suggest that the functional expression of SLC28A2 is similar in sperm of the two species. This interpretation assumes that the transport of adenosine analogs is the rate-limiting step in the pathway that accelerates the beat. Consistent with this interpretation, we find that flagellar responses of human sperm to bicarbonate are near-maximal by 30 sec (Fig. 6), as they are for mouse sperm [3, 6], indicating that responses that follow elevation of the cAMP messenger occur quickly.
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The concentrative nucleoside transporters use the transmembrane Na+ gradient to drive the transport of nucleosides into the cell [23]. The transport of adenosine is blocked by replacing external Na+ with Li+, K+, Cs+, N-methyl-D-glucamine, or choline [28, 29]. As a second test for the involvement of SLC28A in the activation of mouse sperm, we examined the effectiveness of Cl-dAdo in Na+-deficient medium (Fig. 7A). Replacement of external Na+ with Li+ decreased the effectiveness of Cl-dAdo (5.1 ± 0.3 Hz with Li+ vs. 7.6 ± 0.6 Hz with Na+ at 90 sec of Cl-dAdo treatment; P < 0.005). The extrapolated beat doubling time for Cl-dAdo was 44 sec for cells in the presence of Na+ and 144 sec for cells in the presence of Li+ (Fig. 7A). In contrast, removal of Na+ had little effect on the action of 15 mM bicarbonate (Fig. 7B; P > 0.05). Thus, replacement of external Na+ with Li+ does not prevent the activation of rapid motility in general. Cl-dAdo action in human sperm also may be dependent on extracellular Na+. When NaCl was replaced by choline chloride and only 20 mM Na+ remained in the external medium, 120 sec of treatment with Cl-dAdo produced little or no response in human sperm (Schuh, unpublished data).
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The specificity of the response to the substitution of Na+ with Li+ was further examined by measuring cAMP content of mouse sperm treated with HCO3– or Cl-dAdo in Na+-deficient medium (Table 2). Replacement of external Na+ with Li+ decreased the effectiveness of Cl-dAdo at elevating cAMP levels, but did not significantly decrease the effectiveness of HCO3–. Thus, replacement of Na+ with Li+ does not prevent the bicarbonate-evoked increase in cAMP and consequent activation of rapid motility. The analog-selective kinetics of activation and apparent dependence of activation upon external Na+ are consistent with a role of a concentrative nucleoside transporter in the adenosine-mediated acceleration of the beat.
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We have shown that several adenosine analogs and catecholamine agonists accelerate the flagellar beat of mouse and human sperm within tens of seconds. It is unlikely that conventional adenosine or adrenergic G protein-coupled receptors are involved in this signaling pathway. Instead, our results support the hypothesis that adenosine agonists enter the cell through transporters and act at intracellular targets (see a diagrammatic illustration of these hypotheses in supplemental Figure 1 available online at www.biolreprod.org).
Adenosine and Catecholamine Action in Human Sperm
We found a resting beat rate of human sperm (6.5 Hz at room temperature) more than double that of mouse sperm. Analyses of swimming paths by computer-assisted semen analysis have reported "beat cross frequencies" of 12–25 Hz [30, 31]; beat cross frequency is approximately twice the flagellar beat frequency [30]. Tracking free-swimming human sperm, Mortimer et al. reported a flagellar beat of 35 Hz for human sperm examined at 37°C in medium containing human serum albumin and HCO3– [32].
No previous studies have examined the acute actions of adenosine and catecholamines on the flagellar waveform of single human sperm and compared the responses of human and mouse sperm to these agents. In populations of human sperm exposed for minutes to hours with adenosine and analogs [21, 22, 33] or catecholamine agonists [34], past work reported an increase in the percentage of motile sperm. Importantly, we find that similar to their action in mouse sperm, adenosine analogs, catecholamines, and bicarbonate rapidly double the beat frequency of human sperm. These molecules are present in human reproductive fluids, and therefore they may have a role in human sperm capacitation in vivo.
Evidence Against Conventional Adenosine Receptors
Immunoreactivity for adenosine A1 and A2A receptors (ADORA1 and ADORA2A) and binding sites consistent with ADORA2 receptors have been reported for sperm [18, 20, 21]. Transcripts of Adora2a also have been detected in round spermatids [35]. It has been suggested that adenosine A2A receptors may have a functional role in adenosine-mediated stimulation of sperm motility and capacitation [18, 21, 22, 36, 37]. However, others reported that the potency order and structure-activity relationships of various adenosine analogs in sperm indicate a unique receptor or a non-receptor-mediated action [33, 38]. Specifically, adenosine itself did not increase intracellular cAMP levels. Moreover, the most effective stimulator of motility and cAMP elevation in bovine [38] and human [33] sperm was 2'-deoxyadenosine, an adenosine analog with weak activity at adenosine receptors. Further, knockout mice carrying targeted disruptions of Adora1, Adora2a, or Adora3 remain fertile [39].
Using a pharmacological approach, we found that a potent agonist and an antagonist for adenosine receptors were ineffective at stimulating or inhibiting, respectively, the acceleration of beat. It seems unlikely that conventional adenosine receptors are involved in the activation of motility, but a potential role for receptors in later stages of capacitation cannot be excluded by our work.
Evidence Against Conventional Adrenergic Receptors
Several past studies did not detect adrenergic receptors in mature sperm [40–42]. However, immunologic evidence was found recently for several beta and alpha adrenergic receptor subtypes (ADRB1, ADRB2, ADRB3, ADRA2A) in mouse and human sperm, and it was proposed that these receptors function in NE-stimulated capacitation via a G protein-coupled/transmembrane adenylyl cyclase pathway [19]. pharmacological sensitivity profiles of hamster, mouse, and bull sperm capacitation also provide some support for involvement of adrenergic receptors [19, 42–45]. Meizel and Working [45] reported that the l-(–) isomers of ISO, NE, or E were more effective than the d-(+) isomers in promoting hamster sperm capacitation as assessed by spontaneous acrosome reactions, which is consistent with the isomer selectivity of adrenergic receptors in somatic cells [46]. However, in the activation of the flagellar beat we did not find specificities typical for adrenergic receptors. Several β-adrenergic agonists as well as antagonists had significant accelerating actions. This response was not specific to the l-(–) isomers. Thus, it is unlikely that conventional receptors are involved in the catecholamine-mediated acceleration of sperm beat.
Adenosine Action via an Equilibrative Nucleoside Transporter
The SLC29A family of equilibrative nucleoside transporters produce facilitated diffusion that is sensitive to selective inhibitors [23, 47]. Early studies reported that the SLC29A inhibitor dipyridamole (0.01 and 0.1 µM) did not prevent adenosine from increasing the proportion of motile sperm [22] or the proportion of in vitro fertilization [36]. Nanomolar concentrations of NBTI did not displace radiolabled NECA that was bound to putative adenosine receptors on mouse sperm, and it was concluded that NECA was acting at an external site and not entering the cell [37]. These studies suggested that adenosine action in sperm did not involve transporters. Nevertheless, we found that SLC29A inhibitors slow the onset of the acceleration of beat evoked by adenosine analogs, suggesting that an SLC29A may be involved.
Previous work incubated populations of sperm with the inhibitor alone for 20 min or more and then examined the effects of the inhibitor in combination with adenosine several minutes to hours later. These studies did not examine the effects of these inhibitors on the rapid activation of motility within the first seconds of agonist exposure. As these inhibitors are likely to slow rather than completely prevent the transport of all adenosine, it seems likely that after several minutes, adenosine entry into the cell is sufficient for action at intracellular targets and alterations in sperm motility.
Member 1 of SLC29A may mediate the entry of adenosine analogs into sperm. The two best-characterized mammalian members of the equilibrative nucleoside transporter family, SLC29A1 and SLC29A2, have similar broad selectivities for purine and pyrimidine nucleosides. Although spermatogenic cells contain transcripts for members 1 and 3 of Slc29a [35], only member 1 is located on the plasma membrane and is significantly inhibited by the transport inhibitors NBTI and dipyridamole [23, 48]. Therefore, we propose that adenosine may act via SLC29A1 in sperm. At 100 nM, the SLC29A1 inhibitor NBTI completely inhibits adenosine transport in bovine chromaffin and several other somatic cells [49]. Micromolar dipyridamole also strongly inhibits SLC29A1-mediated transport of nucleosides [50]. Because the onset of the Cl-dAdo-mediated acceleration of beat was slowed approximately two thirds by these inhibitors, we reasoned that other transporters may also contribute to adenosine action in sperm.
Adenosine Action via a Concentrative Nucleoside Transporter
Many somatic cells use both equilibrative and concentrative nucleoside transporters to transport nucleosides [23, 51]. Gene array studies on the developing mouse testis and spermatogenic cells found a small signal for member 2 of the concentrative nucleoside transporter family, Slc28a2 [35]. Because the adenosine analogs we used are purine nucleosides, which typically enter somatic cells via SLC28A2 [23, 27], we postulated that SLC28A2 could be an additional candidate transporter for entry of adenosine analogs into sperm. The species differences in the kinetics of activation mediated by adenosine analogs and the requirement for external Na+ are consistent with a contribution of SLC28A2 in this signaling pathway in sperm. Similar to other cells that express multiple types of transporters, a redundancy of mechanisms facilitating adenosine transport may reflect the importance of adenosine signaling in sperm.
The ribose 3'-hydroxyl group of nucleosides is an important determinant for interaction with and transport via concentrative nucleoside transporters [52], whereas the 2'- and 3'-hydroxyls are important for interaction with adenosine receptors [53, 54]. Several of the adenosine analogs that are effective in sperm lack the 2'-hydroxyl and are weak agonists of adenosine receptors. All of them contain the 3'-hydroxyl. Further, the analog Cl-dAdo has long been used in the chemotherapy of hematologic malignancies, with its main action at intracellular targets [55]. Conversely, the potent adenosine A2 receptor agonist, CGS21680, that we found ineffective in sperm, is not likely to use nucleoside transporters. These considerations favor involvement of transporters rather than receptors in the action of adenosine analogs.
As adenosine- and adrenergic-mediated signaling pathways underlying sperm activation seem similar [6] and the transcripts of several catecholamine transporters, including organic cation transporters, monocarboxylic acid transporters, and equilibrative nucleoside transporter 4, are in spermatogenic cells [35, 56], we suggest that the catecholamines may also use specific transporters to enter the cell. Future pharmacological and molecular studies could examine the putative role of specific transporters in the catecholamine-signaled activation of sperm.
In conclusion, several adenosine and adrenergic agonists stimulate the beat of sperm of both mouse and human. Our past and present findings suggest that these small molecules act in a novel signaling pathway that is mediated by cAMP and requires SACY, protein kinase A, and external Ca2+, and for adenosine analogs, may involve transport into the cell through transporters, rather than action at a conventional cell-surface receptor. The intracellular targets that couple adenosine and catecholamines to the cAMP rise are unknown. It is possible that the agonists act by stimulating the SACY enzyme directly or indirectly, or by inhibiting PDE to increase accumulation of SACY-generated cAMP. Understanding the signaling events of mammalian sperm activation and, specifically, how sperm respond to putative external chemical signals en route to the egg should provide insight into gamete physiology and the complex process of capacitation. These agonists and the proteins involved in their signaling pathways may hold promise for contraception and for treatment of infertility.
ACKNOWLEDGMENTS
We thank Dr. John Amory and coworkers for recruiting human subjects and generously providing the human semen samples. We thank Eric Nguyen for creating ImageJ plug-ins for enhancement of waveform images and for assistance with image processing. We also thank Blake Nichols and Dr. G. Stanley McKnight for providing the EIA kit and the necessary equipment for performing the cAMP experiments.
FOOTNOTES
1Supported by National Institute of Child Health & Human Development/National Institutes of Health (NIH) through cooperative agreement [U54 (HD12629)] as part of the Specialized Cooperative Centers Program in Reproduction and Infertility Research. S.M.S. was supported in part by NIH grant 5T32 HD07183. 
Correspondence: 2Donner F. Babcock, Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290. FAX: 206 685 3191; e-mail: donner{at}u.washington.edu
Received: 3 May 2007.
First decision: 3 June 2007.
Accepted: 15 August 2007.
REFERENCES
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