Hum. Reprod. Advance Access originally published online on December 7, 2006
Human Reproduction 2007 22(3):756-765; doi:10.1093/humrep/del454
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Cathine, an amphetamine-related compound, acts on mammalian spermatozoa via 
1- and 
2A-adrenergic receptors in a capacitation state-dependent manner
1 Division of Reproduction and Endocrinology, School of Biomedical and Health Sciences, King's College London, London, UK
2 To whom correspondence should be addressed at: Division of Reproduction and Endocrinology, School of Biomedical and Health Sciences, King's College London, London SE11UL, UKTel: 44 20 7848 6272; Fax: 44 20 7848 6220; E-mail: lynn.fraser{at}kcl.ac.uk
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Abstract |
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BACKGROUND: Mammalian spermatozoa have been shown to have
1,2,3- and 
2A-adrenergic receptors, the former functioning only in uncapacitated spermatozoa and the latter only in capacitated cells. Cathine, an amphetamine-related metabolite of a compound found in Catha edulis leaves, accelerates capacitation and inhibits spontaneous acrosome loss by regulating cAMP production. This study tested the hypothesis that adrenergic receptors are involved in these responses.
METHODS: Uncapacitated and capacitated mouse sperm suspensions were incubated with cathine ± specific antagonists for 2- and -adrenergic receptors for 35 min, then assessed using chlortetracycline fluorescence. Reversibility of receptor accessibility was assessed by depleting suspensions of endogenous decapacitation factor (DF) and then adding crude DF with/without cathine and antagonists. Effects on tyrosine phosphorylation and calcium requirements for both ligand binding and biological responses were also evaluated.
RESULTS: Cathine's acceleration of capacitation was blocked by a 1-antagonist, whereas an 2-antagonist blocked inhibition of acrosome reactions. Cathine accelerated capacitation in decapacitated cells, a response inhibited by a 1-antagonist; cathine also stimulated tyrosine phosphorylation. Although calcium was not required for binding, it was needed for responses.
CONCLUSIONS: Cathine acts at 1-adrenergic receptors in uncapacitated spermatozoa and at 2A-receptors in capacitated cells; biological activity requires calcium but binding does not. Adrenergic receptor-binding sites can be made reversibly accessible/inaccessible by changing the capacitation state of spermatozoa. These results suggest that amphetamine-related compounds might enhance chances of fertilization in vivo.
Key words: amphetamine/capacitation/fertility/membrane-associated adenylyl cyclase/tyrosine phosphorylation
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Introduction |
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Cathine and norephedrine, phenylpropanolamines (PPAs) structurally related to amphetamine, are stable metabolites of cathinone, a naturally occurring alkaloid found in leaves of the shrub Catha edulis (khat), which are often chewed recreationally. Cathinone has euphoric, stimulatory properties, reflecting its structural similarities with amphetamine (Brenneisen et al., 1990
; Kalix, 1992). Both PPAs have been shown to affect mammalian sperm function in biologically important ways (Adeoya-Osiguwa and Fraser, 2005). When mammalian spermatozoa leave the male reproductive tract, they are not yet able to fertilize; they must undergo further post-release maturational events, collectively referred to as ‘capacitation’, that confer on them the ability to fertilize an oocyte (Austin, 1952). Capacitation requires a variable amount of time, depending on the species. Using chlortetracycline (CTC) fluorescence analysis, we have shown that cathine and norephedrine significantly accelerate capacitation in both uncapacitated mouse and human spermatozoa; they also accelerate in vitro fertilization and inhibit spontaneous acrosome loss in mouse spermatozoa. Pertussis toxin blocked effects on capacitated cells, suggesting involvement of inhibitory G proteins. Finally, cathine stimulated cAMP production in uncapacitated mouse spermatozoa but inhibited it in capacitated cells (Adeoya-Osiguwa and Fraser, 2005).
Because some studies on somatic cell systems had suggested that PPAs might act by binding to adrenergic receptors (e.g. Rothman et al., 2003), both adrenaline and noradrenaline were tested and found to elicit the same responses as PPAs. Uncapacitated suspensions pre-incubated for only 15 min in the presence of adrenaline or cathine were significantly more fertile than untreated controls when analysed after just 75 min co-incubation with oocytes, indicating stimulation of both hyperactivation and changes in the sperm head required for successful fertilization. Furthermore, adrenaline, like cathine, stimulated cAMP production quickly and significantly in uncapacitated spermatozoa but inhibited it in capacitated cells, consistent with mammalian spermatozoa having adrenergic receptors (Adeoya-Osiguwa and Fraser, 2005).
Adrenergic receptors are G protein-coupled receptors (GPCRs) and several subtypes have been shown to modulate the activity of membrane-associated adenylyl cyclases (mACs) in somatic cells (Watling, 2001). A number of mAC isoforms have been identified in mammalian spermatozoa (Baxendale and Fraser, 2003; Wade et al., 2003) and evidence from studies on several other GPCRs suggests that the mACs are functional (Fraser et al., 2003). Mammalian spermatozoa also have a soluble isoform of AC (sAC; activated by HCO3– and Ca2+) that appears to play an important role in sperm function (Buck et al., 1999). However, this AC appears not to be regulated by G proteins (Wuttke et al., 2001) and so is unlikely to be involved in responses to cathine. Since the culture medium used in our earlier study (Adeoya-Osiguwa and Fraser, 2005) contained both HCO3– and Ca2+, sAC would have been functioning; even so, cathine and adrenaline were able to cause significant changes in cAMP production, consistent with mAC involvement.
For the last three decades, there has been interest in the possible presence of adrenergic receptors on mammalian spermatozoa. Several studies showed that adrenal gland extracts, known to contain catecholamines such as adrenaline, and individual catecholamines had positive effects on hamster sperm motility (either general or hyperactivated), capacitation, acrosome reactions and fertilizing ability in vitro (e.g., Bavister et al., 1976; Cornett and Meizel, 1978; Bavister et al., 1979; Cornett et al., 1979; Meizel and Working, 1980; Bize and Santander, 1985). However, the concentration of catecholamines used was quite high, commonly about 20–50 µmol/l, and may have produced non-physiological responses. More recently, Way et al. (2001) reported finding low-to-moderate nanomolar concentrations of noradrenaline in bovine oviductal fluid. In subsequent investigations of bovine sperm responses to noradrenaline and adrenaline, Way and Killian (2002) found that only noradrenaline appeared to affect the cells, with low concentrations stimulating but high concentrations inhibiting both capacitation and acrosome reactions. The authors suggested the possible presence of two populations of receptors but had no direct evidence to support this. Given the general lack of convincing evidence, Meizel (2004) recently concluded that mammalian spermatozoa are unlikely to have adrenergic receptors.
Because the results with cathine, norephedrine, adrenaline and noradrenaline all suggested that adrenergic receptors might be present, it seemed reasonable to look for them, using specific antibodies, agonists and antagonists for different receptor subtypes that are now available. The focus was on -receptors, known to stimulate mAC activity in somatic cells, and 2-receptors, known to interact with pertussis toxin-sensitive inhibitory G subunits to inhibit mAC activity in somatic cells; in contrast, 1-receptors usually stimulate phospholipase C via pertussis toxin-insensitive G subunits (Watling, 2001).
Western blotting and immunolocalization analysis provided strong evidence that mouse and human spermatozoa have 2A-, 1-, 2- and 3-adrenergic receptors; all are located on both the head and the flagellum and appear to be functional (Adeoya-Osiguwa et al., 2006). All three -receptor populations caused an acceleration of capacitation in uncapacitated cells, but the magnitude of response to specific agonists differed, with 1 > 2 > 3. -Receptor agonists had no effect on capacitated suspensions, whereas a specific 2-receptor agonist acted only on capacitated cells, causing inhibition of spontaneous acrosome loss. Thus, it appears that the -receptors only function during the early stages of capacitation, whereas 2A-receptors only function in capacitated cells.
These findings are consistent with the evidence that cathine and adrenaline stimulate cAMP production in uncapacitated suspensions and then inhibit it in capacitated ones (Adeoya-Osiguwa and Fraser, 2005). Inclusion of 2',5'-dideoxyadenosine, a specific inhibitor of mACs but not sAC (Johnson et al., 1997), blocked responses to noradrenaline, supporting our hypothesis that adrenergic receptors in spermatozoa regulate mAC activity. A recent study addressing mechanisms involved in stimulating mouse sperm motility claimed to have shown that signalling pathways for adenosine and catecholamine agonists involving GPCRs and mACs are not functional in spermatozoa (Schuh et al., 2006). However, a medium lacking HCO3– was used to test sperm responses to agonists; such a medium will not support capacitation and so is non-physiological. In addition, 2-chloro-2'-deoxyadenosine, used as an adenosine agonist, is not recognized as a potent agonist acting at adenosine GPCRs (Watling, 2001); indeed, evidence suggests that it may act intracellularly rather than via external receptors (Ceruti et al., 2000). The weight of current evidence favors an important role for GPCRs in regulating cAMP production during capacitation so that spermatozoa switch on quickly and then retain fertilizing potential by maintaining an intact acrosome until they interact with unfertilized oocytes (Fraser et al., 2003).
The present study was designed to test the hypothesis that cathine, like adrenaline and noradrenaline, acts on mammalian spermatozoa via adrenergic receptors. Antagonists were used to determine whether cathine binds to specific receptors and, if so, which ones. Binding studies were carried out to determine whether cathine binds to the same sites as noradrenaline and the calcium requirements for both binding and biological responses were evaluated. Finally, we tested the hypothesis that, by stimulating cAMP production in uncapacitated spermatozoa, cathine enhances protein tyrosine phosphorylation.
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Materials and methods |
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Media and reagents
A modified Tyrode's medium (Fraser, 1993
) containing 1.8 mmol/l CaCl2, 25 mM NaHCO3 and 4 mg/ml bovine serum albumin (BSA) was used. Unless otherwise stated, all reagents were purchased from Sigma (Poole, Dorset, UK). Fresh stock solutions were prepared daily at 5 mmol/l and diluted using standard culture medium. The solvents used to prepare stock solutions were: DL-noradrenaline hydrochloride, procaterol hydrochloride and SKF 86466 - culture media; ICI 118551 - absolute ethanol; CGP 20712A and UK 14304 - DMSO. All stock solutions were then diluted 1000-fold or more in medium and used at 1/50; in our experience, the final concentrations of these non-aqueous solvents have no detectable effect on sperm function. Agonist and antagonist concentrations used here are those found to be optimal in our earlier investigation (Adeoya-Osiguwa et al., 2006).
Sperm suspension preparation
For each replicate experiment, cauda epididymal mouse spermatozoa from at least two or three mature TO males (Harlan, Bicester, UK), depending on the amount of suspension required, were released into sterile plastic dishes (Nunc, Roskilde, Denmark) containing modified Tyrode's medium (
0.8 ml per pair of epididymides). In experiments using uncapacitated cells, suspensions were allowed to disperse for 5 min on a warming tray and then filtered through short columns of Sephadex G-25 [medium grade; GE Healthcare (formerly Amersham Biosciences), Little Chalfont, UK], pre-equilibrated with medium, to remove non-motile cells. Filtrates were pooled, assessed to determine that motility was satisfactory (90% motile), and then immediately treated as detailed in the Results.
For capacitated cells, spermatozoa were released into modified Tyrode's medium and allowed to disperse; after assessment for motility, suspensions were covered with medium-equilibrated autoclaved liquid paraffin (Boots, Nottingham, UK) and incubated in a gas phase of 5% CO2: 5% O2: 90% N2 at 37°C for a minimum of 90 min. Suspensions were then filtered as described earlier. This protocol has been shown to consistently produce capacitated suspensions as evidenced by CTC analysis (e.g. Fraser et al., 1997), in vitro fertilization (e.g. Fraser et al., 1997) and protein tyrosine phosphorylation analysis (Adeoya-Osiguwa and Fraser, 2000).
CTC fluorescence analysis
The CTC fluorescence assay (Green et al., 1994) was used to assess sperm functional state. Assessments were carried out on an Olympus BX41 microscope [Olympus Optical Co. (UK) Ltd, London, UK] equipped with phase contrast and epifluorescent optics; the U-MWBV2 fluorescence cube (wide blue–violet) is the one suitable for CTC. In each replicate, 100 spermatozoa in each treatment sample were classified into three staining patterns: F, with fluorescence over the entire head, characteristic of uncapacitated, acrosome-intact spermatozoa; B, with a fluorescence-free band in the post-acrosomal region, characteristic of capacitated, acrosome-intact spermatozoa; AR, with dull or absent fluorescence over the entire head, characteristic of acrosome-reacted spermatozoa.
3H-noradrenaline-binding studies
Radioligand-binding studies were performed using 3H-noradrenline (36 Ci/mmol; GE Healthcare); preliminary investigations indicated that 100 nmol/l was the optimal concentration to use in experiments. Sperm suspensions were prepared and treated first with unlabelled reagents and then 3H-noradrenline, as described in the Results. Binding was terminated by filtering triplicate 100 µl aliquots of each sample; each filter was then washed three times with 5 ml ice-cold 50 mmol/l Tris–HCl buffer, pH 7.5, containing 2 mM EDTA. The amount of bound 3H-noradrenaline was determined by adding 2 ml scintillation fluid [Optiphase Gold; PerkinElmer LAS (UK) Ltd, Beaconsfield, UK] to each air-dried filter and then counting. Total counts per sample were determined by adding 5 µl of unfiltered reaction mixture to a filter paper which was dried and then counted. Counts obtained for non-specific binding were subtracted from each treatment group; specific binding was then corrected for the number of spermatozoa in each suspension used in order to obtain fmol 3H-noradrenaline bound per 1 x 107 cells.
Electrophoresis, western blotting and detection of tyrosine phosphoproteins
Sperm suspensions were released into complete medium, allowed to disperse for 5 min and filtered; 50 mmol/l sodium orthovanadate (phosphatase inhibitor) was added to give a final concentration of 100 µmol/l. Three aliquots from each replicate suspension were treated experimentally as described in the Results; treatment was terminated by transferring 400 µl of each sperm suspension into tubes containing reagents to give final concentrations of 4 mmol/l EDTA, 0.2 µg/ml leupeptin and 400 µg/ml trypsin inhibitor. Care was taken to mix suspensions well at each step to ensure that similar numbers of spermatozoa would be present in each sample used for electrophoresis. Samples were frozen in liquid N2, thawed at room temperature and centrifuged at 10 000g for 6 min at 4°C to pellet the cells. Spermatozoa were then resuspended to 3 x 107 cells/ml with PBS containing 100 µmol/l sodium orthovanadate, 4 mM EDTA, 0.2 µg/ml leupeptin and 400 µg/ml trypsin inhibitor. Each sample was solubilized in loading buffer (Gibbons et al., 2005), denatured in boiling water for 7 min and stored in aliquots at –20°C until required.
To visualize proteins, 7.5 µl of each sample (in duplicate) was resolved on 10% linear polyacrylamide sodium dodecyl sulphate gels prepared using protocols from National Diagnostics (Hull, UK); rainbow recombinant standards were used as molecular weight markers (GE Healthcare). After electrophoresis, proteins were electro-transferred onto polyvinylidene difluoride (PVDF) membranes, as described by Gibbons et al. (2005). Membranes were blocked for 1 h at room temperature in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 (TBS-T) + 1% BSA, overlaid with the primary antibody (PY20-HRP conjugate; product no. RPN2220, GE Healthcare) diluted 1:10 000 in TBS-T and then incubated for 1 h at room temperature with gentle agitation. PVDF membranes were then washed with copious TBS-T (1 x 5 min, 3 x 5 min) prior to enhanced chemilumnescence (ECL) detection, as described in the protocol from the Amersham ECL plus kit (RPN2132; GE Healthcare). Membranes were vacuum-sealed, placed in an X-ray film cassette and exposed to Hyperfilm ECL (GE Healthcare) for 30–45 s.
Statistical analysis
CTC data were analysed using Cochran's modification of the 
2 test, a test which compares responses within each replicate and then sums the values (Snedecor and Cochran, 1980). In order to obtain a statistically significant difference, responses must be consistent and the difference in responses between the experimental and control values must be sufficiently large in each replicate. Results from the binding studies were analysed using a paired t-test (SigmaStat; Jandell Scientific International, Chicago, IL, USA).
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Results |
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Series I: Does cathine elicit responses in uncapacitated spermatozoa via
-adrenergic receptors?If cathine acts via adrenergic receptors to alter cAMP production, we hypothesized that it would produce its effects on uncapacitated cells via
-receptors, known to interact with mACs to stimulate cAMP production (Watling, 2001). Although mouse and human spermatozoa have 1-, 2- and 3-receptors, results obtained with specific agonists and antagonists suggested that the 3-receptors were the least active (Adeoya-Osiguwa et al., 2006) and so the study concentrated on 1- and 2-receptors.
Uncapacitated suspensions were prepared as described earlier, divided into aliquots and treated with agonists at 100 nmol/l and antagonists at concentrations used in the study of Adeoya-Osiguwa et al. (2006). Treatments were: (i) nothing (control); (ii) cathine; (iii) cathine + 100 nmol/l CGP 20712A (1-antagonist); (iv) cathine + 10 nmol/l ICI 118551 (2-antagonist); (v) noradrenaline (1-agonist); (vi) noradrenaline + CGP 20712A; (vii) 100 nmol/l procaterol (2-agonist); (viii) procaterol + ICI 118551. After 35 min, samples were stained with CTC and fixed; slides were prepared and evaluated. We have found that this length of incubation is sufficient to detect whether or not a treatment has a significant effect on either uncapacitated or capacitated cells. At the end of treatment, samples were quickly evaluated for general motility to ensure that treatment had not adversely affected motility; in all samples, motility was still high (
80%). Three replicate experiments were carried out (n = 3).
As shown earlier (Adeoya-Osiguwa and Fraser, 2005; Adeoya-Osiguwa et al., 2006), cathine, noradrenaline and procaterol all significantly accelerated capacitation and CGP 20712A effectively blocked responses to noradrenaline, and ICI 118551 blocked responses to procaterol. When cathine was tested with the two -receptor antagonists, only CGP was able to block the response (Figure 1); ICI had no detectable interference with cathine's action. This suggests that cathine binds only to 1-receptors on uncapacitated cells.
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), B pattern (
) and AR pattern (
) of CTC fluorescence. ****P < 0.001 compared with the untreated controls (Con).Series II: Does cathine elicit responses in capacitated spermatozoa via
2-adrenergic receptors?To test the hypothesis that cathine works on capacitated cells via
2-receptors, known to interact with mACs to inhibit cAMP production (Watling, 2001), suspensions were prepared, pre-incubated to allow capacitation and then filtered as described earlier. Before starting experimental treatment, an aliquot of the filtered capacitated suspension (control post-filtration) was immediately stained with CTC and fixed in order to provide information on the proportions of F, B and AR pattern cells present prior to treatment. The rest of the suspension was divided into aliquots and treated as follows, with cathine being used at 100 nmol/l: (i) nothing (control); (ii) cathine; (iii) cathine + 10 nmol/l SKF 86466 (2-antagonist); (iv) 1 nmol/l UK 14304 (2-agonist); (v) UK 14304+SKF 86466. After 35 min, samples were stained with CTC and fixed; slides were prepared and evaluated (n = 3). Motility was still high in all unfixed samples at the end of treatment.
As shown in Figure 2, a large number of spermatozoa in the untreated control suspensions underwent spontaneous acrosome reactions during the 35 min incubation following filtration. Consistent with our earlier study (Adeoya-Osiguwa et al., 2006), UK 14304 blocked this loss; inclusion of the antagonist SKF 86466 interfered with responses to the agonist so that the proportion of acrosome-reacted cells was essentially the same as that seen in the untreated controls. Cathine also blocked acrosome loss and the 2-antagonist abolished this response, thus confirming our hypothesis that cathine acts on capacitated spermatozoa via 2-receptors.
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Series III: Does decapacitation cause changes in the availability of adrenergic receptor-binding sites?
Adeoya-Osiguwa et al. (2006)
reported that the 2-agonist UK 14304 had no effect on uncapacitated spermatozoa, whereas the 2-agonist procaterol had no effect on capacitated cells, even though both were able to significantly affect cells in the opposite capacitation state. This suggests that the adrenergic receptors function in a capacitation state-dependent manner, with accessibility of their binding sites being changeable. We wished to test the hypothesis that accessibility of binding sites is reversible. To do this, we depleted suspensions of endogenous DF by gentle centrifugation to promote rapid capacitation (Fraser, 1984) and then treated them with crude DF ± cathine, CGP 20712A and SKF 86466 at the concentrations used in earlier experimental series.
Uncapacitated sperm suspensions were prepared and incubated for 25 min. An aliquot was removed, diluted 1:1 with medium and centrifuged at 4500 g for 8 min to pellet; the supernatant, containing crude DF, was removed and kept on ice until just prior to use. The remaining suspension was filtered and then divided into aliquots. All but one of these aliquots was centrifuged at 1100 g to deplete endogenous DF. Supernatant was removed and cells were resuspended in either control medium or crude DF (using half the original volume to compensate for loss of spermatozoa not pelleted by the gentle spin); other reagents were then added. The final sperm concentration was 1 + 2 x 107 cells/ml. Treatments of the spun aliquots included: (i) none (DF-depleted = spun control); (ii) crude DF; (iii) crude DF + cathine; (iv) crude DF + cathine + CGP 20712A; (v) crude DF + cathine+ SKF 86466. The uncentrifuged sample served as the unspun control, which should show a typical uncapacitated profile of CTC patterns. All samples were incubated for 35 min and then analysed (n = 3).
Figure 3 demonstrates that the majority of cells in the unspun control samples displayed the uncapacitated F pattern, whereas the capacitated B pattern predominated in the spun controls (capacitated by DF-depletion); 2-receptors would be available in those capacitated cells (Figure 2). The data for the spun controls demonstrate why mouse spermatozoa should not be subjected routinely to centrifugation: centrifugation profoundly alters the physiology of the cells. Addition of crude DF to the DF-depleted cells resulted in decapacitation, with the CTC profile resembling that seen in the unspun uncapacitated controls. Cathine added to the decapacitated suspensions promoted re-capacitation, but to which population of adrenergic receptors was cathine binding? Since CGP 20712A blocked the responses to cathine and SKF 86466 had no effect, cathine was acting at 1- rather than at 2-adrenergic receptors. Thus, decapacitation results in restoration of 1-receptor-binding site accessibility, confirming the hypothesis that the receptor-binding sites can be made reversibly accessible/inaccessible.
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Series IV: Is calcium required for ligands to bind to adrenergic receptors and/or for ligands to elicit biological responses?
Since the CTC results indicated that both cathine and noradrenaline bind to
1-receptors, we tested the hypothesis that cathine would competitively inhibit 3H-noradrenaline binding to intact spermatozoa; we also investigated whether calcium is required for binding and/or functional responses. Preliminary binding experiments used sperm suspensions prepared in complete, Ca2+-containing, medium. Results (data not shown) indicated that labelled noradrenaline did bind to cells and that the inclusion of cathine reduced the amount of bound noradrenaline, but values were not consistent. We interpreted this to indicate that, in complete medium, spermatozoa are undergoing the physiological changes associated with capacitation and the inclusion of noradrenaline and/or cathine will cause them to change even more rapidly than in medium alone. Therefore, although -receptor-binding sites will be available initially, after some minutes, 2-binding sites will begin to be available and so ligands could be binding to both populations of receptors. Therefore, binding was investigated using suspensions prepared in Ca2+-deficient medium; these conditions prevent completion of capacitation (Fraser, 1982) and so only -receptors should be available for binding.
Sperm suspensions were prepared in Ca2+-deficient medium (no added CaCl2), allowed to disperse and then filtered. Prior to the addition of 3H-noradrenline, suspensions were divided into five aliquots and treated with: (i) nothing (control); (ii) CGP 20712A (1-antagonist); (iii) 100 nmol/l cathine; (iv) 1000 nmol/l cathine. The fifth aliquot, to which 100 µmol/l unlabelled noradrenaline was added, was used to determine non-specific binding as described in Materials and Methods. After incubation for 10 min at 37°C, 3H-noradrenline was added to all tubes to give a final concentration of 100 nmol/; suspensions were incubated for a further 5 min and then prepared for analysis (n = 6). As seen in Figure 4, the binding of 3H-noradrenaline was significantly lower in the presence of CGP 20712A and cathine, the magnitude of inhibition being similar with both compounds. These results indicate that binding to adrenergic receptors does not require the presence of Ca2+.
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To test whether physiological responses to cathine and noradrenaline require the presence of extracellular Ca2+, suspensions were prepared in Ca2+-deficient medium and then divided into two; to one aliquot, a concentrated stock solution of CaCl2 was added to give a final concentration of 1.8 mmol/l. Each of these suspensions was then divided into aliquots and treated with: (i) nothing (control); (ii) cathine; (iii) noradrenaline. After 35 min, samples were stained with CTC and fixed; slides were prepared and evaluated. As shown in Figure 5, both cathine and noradrenaline significantly accelerated capacitation in Ca2+-containing medium, but there was no detectable response in the Ca2+-deficient medium.
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Series V: Do cathine and noradrenaline stimulate protein tyrosine phosphorylation in uncapacitated spermatozoa?
Our earlier investigation of responses to cathine demonstrated that the acceleration of capacitation seen in response to cathine, adrenaline and noradrenaline using CTC analysis correlated with a significant increase in cAMP production (Adeoya-Osiguwa and Fraser, 2005
). We therefore hypothesized that such responses would also cause an increase in protein tyrosine phosphorylation.
Filtered uncapacitated sperm suspensions containing 100 µmol/l sodium orthovanadate were mixed well, divided into three aliquots and treated with: (i) nothing (control); (ii) 100 nmol/l noradrenaline; (iii) 100 nmol/l cathine. Samples were again mixed gently, gassed for 15 s and incubated for 10 min at 37°C and then evaluated using gel electrophoresis and western blotting as described in Materials and Methods. Three different samples were evaluated, with each being used for two or three replicate gels; Figure 6 shows a representative result. As can be seen, both noradrenaline and cathine did stimulate tyrosine phosphorylation during the 10 min treatment interval, compared with the untreated control. In particular, tyrosine phosphoproteins of 35, 42, 48, 56, 75–82 and 116 kDa (denoted by asterisks) appear to be more heavily phosphorylated in the treated than in the control samples.
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The results obtained in this study firmly support the hypothesis that mammalian sperm responses to cathine involve both
- and 2-adrenergic receptors. Using receptor-specific antagonists, cathine was shown to act on uncapacitated cells by binding only to 1-receptors, whereas it acts via 2-receptors in capacitated cells. This capacitation state-dependence of receptor involvement appears to reflect restricted accessibility of receptor-binding sites, -sites only being accessible in uncapacitated cells and 2-sites only being accessible in capacitated cells. This accessibility can be reversed by experimentally changing the capacitation state: the addition of exogenous DF to cells capacitated by DF-depletion resulted in reversion to the uncapacitated state, with 1-receptors rather than 2-receptors being available for binding.
In the 3H-noradrenaline-binding studies, both cathine and CGP 20712A significantly interfered with, but did not completely inhibit, the binding of the labelled noradrenaline. This presumably reflects the differences in specificity, with cathine and CGP only binding to 1-receptors, whereas noradrenaline can bind to 1-, 2- and 3-receptors, (Watling, 2001). The magnitude of inhibition was very similar for both ligands, suggesting that they have similar binding affinities. Although at least three subtypes of 2-receptors have been identified in somatic cells, Adeoya-Osiguwa et al. (2006) found immunohistochemical evidence for only 2A-receptors. Given the lack of good subtype-specific agonists for 2A-receptors, receptor type-specific agonists and antagonists have been used in our studies. The 2-antagonist SKF 86466 blocked responses to cathine in capacitated cells, indicating that cathine can bind to the 2A-adrenergic receptors shown to be present on both mouse and human spermatozoa.
The fact that binding does not require the presence of extracellular Ca2+, but biological responses do, suggests that Ca2+ is required for a step downstream of binding to the receptor. As mentioned earlier, adrenergic receptors are GPCRs that can act upon mACs and many mAC isoforms are stimulated by Ca2+ (Hanoune and Defer, 2001). In studies on fertilization-promoting peptide (FPP), a similar Ca2+ requirement for functional responses (Green et al., 1996) but not for binding (Adeoya-Osiguwa et al., 1998) has been demonstrated. In contrast, calcitonin, which binds to specific calcitonin GPCRs that modulate mAC/cAMP, significantly stimulated both capacitation (CTC analysis) and cAMP production in suspensions prepared in Ca2+-deficient medium, although the responses were lower than in complete medium. When Ca2+ was added to these suspensions to allow the acrosome reaction, they proved to be significantly more fertile than control suspensions (Adeoya-Osiguwa and Fraser, 2003). This was a surprising result, given that both FPP and calcitonin elicit the same responses, acceleration of capacitation followed by inhibition of the acrosome reaction, by regulating cAMP production. The most plausible explanation is that the FPP and calcitonin-signalling pathways involve two different mACs, one of which is able to function with much less Ca2+ than the other. Our current evidence would suggest that responses to amphetamine-related compounds might involve the same mAC isoform regulated by FPP, but further investigation is needed. The simplest pathway, shown in Figure 7, would have the two different adrenergic receptor populations regulating the activity of a single mAC via different G proteins, but involvement of two different mAC isoforms cannot be ruled out at present.
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Because cathine and adrenaline stimulate cAMP production in uncapacitated spermatozoa, we hypothesized that responses downstream of cAMP would include stimulation of protein tyrosine phosphorylation (Visconti et al., 1995
). Indeed, we have already demonstrated that FPP, adenosine and CGS 21680 (a specific agonist acting on stimulatory adenosine receptors), all of which elicit similar responses in uncapacitated spermatozoa as cathine, adrenaline and noradrenaline, stimulate the phosphorylation of a range of tyrosine phosphoproteins (Adeoya-Osiguwa and Fraser, 2000). In the present study, cathine and noradrenaline also enhanced tyrosine phosphorylation in several proteins; those showing the greatest increase in phosphorylation when compared with the untreated controls were 35, 42, 48, 56, 75–82 and 116 kDa. Proteins of similar sizes were also noted in the studies of Adeoya-Osiguwa and Fraser (2000) and Mededovic and Fraser (2004), the latter investigating responses to angiotensin II.
Both the present study and the one identifying adrenergic receptors on spermatozoa (Adeoya-Osiguwa et al., 2006) provide evidence that these receptors function in a capacitation state-dependent manner, with -receptors being accessible only on spermatozoa that have not completed capacitation and 2A-receptors being accessible only on capacitated cells. This is not unique to adrenergic receptors. Mammalian spermatozoa also have two populations of adenosine receptors with similar restrictions on their function: stimulatory A2A receptors only function during capacitation, whereas inhibitory A1 receptors only function in capacitated cells (Fraser and Adeoya-Osiguwa, 1999; Adeoya-Osiguwa and Fraser, 2002). This appears to be due to changes in receptor conformation that alter binding-site accessibility, with such alterations being reversible: addition of decapacitation factor (DF) to capacitated suspensions switched off the A1 and switched on the A2A adenosine receptors, as shown by CTC and cAMP analyses. These functional changes correlated with reversible changes in the intensity of fluorescence during immunolocalization, presumably reflecting conformational changes in the receptors (Adeoya-Osiguwa and Fraser, 2002).
To test the hypothesis that a similar mechanism controls adrenergic receptors, the capacitation state of spermatozoa was deliberately altered by depleting cells of endogenous DF to accelerate capacitation of cells experimentally. When resuspended in fresh medium and incubated for a short time, suspensions had the CTC profile associated with capacitated cells but when resuspended in crude DF, they had the CTC profile of uncapacitated cells. The DF-depleted capacitated cells would have 2A-adrenergic receptors accessible, but we hypothesized that decapacitating those cells would switch off the 2A- and switch on the 1-adrenergic receptors. Indeed, addition of cathine to the decapacitated suspensions accelerated re-capacitation and the use of specific receptor antagonists confirmed that cathine was acting via 1-rather than 2A-receptors.
What is the mechanism for reversibly altering availability of receptor-binding sites? We recently identified a decapacitation factor receptor (DF-R) as being phosphatidylethanolamine-binding protein 1 (PEBP 1; Gibbons et al., 2005). A specific antiserum to DF-R/PEBP 1 showed the protein to be present in the same regions of both uncapacitated and capacitated spermatozoa (acrosomal cap, post-acrosomal region and flagellum). However, the intensity of fluorescence in different sperm regions was capacitation state-dependent and reversible, possibly reflecting conformational changes in the DF-R caused by binding or loss of DF. Because the presence/absence of DF also alters accessibility of ligand-binding sites on GPCRs, such as adenosine and adrenergic receptors, we have suggested that changes in DF-R conformation may promote significant alterations in the lipid architecture of the sperm plasma membrane that then alter the functionality of many membrane-associated proteins, including GPCRs.
Although capacitation has long been considered to be reversible (Bedford and Chang, 1962), the molecular basis of reversibility has been unknown. We think that our hypothesis is plausible, especially in light of recent data, indicating that capacitation involves important changes in membrane lipid architecture (e.g. Cross, 2004; van Gestel et al., 2005). The rapid reversibility of receptor function caused by the addition of DF to capacitated cells is consistent with an alteration of membrane structure that leads to conformational and functional changes in the GPCRs and the pathways they control. Under normal circumstances, capacitation takes hours rather than minutes, reflecting the gradual loss or destruction of DF. The ability of various GPCR ligands (e.g. cathine, noradrenaline, FPP, adenosine) to accelerate capacitation in both uncapacitated spermatozoa (endogenous DF present) and decapacitated spermatozoa (exogenous DF present) presumably indicates that their activation of receptors results in accelerated DF loss from the sperm plasma membrane. The mechanisms involved in DF loss have yet to be identified.
In conclusion, cathine is just one of several compounds structurally related to amphetamine that can act via adrenergic receptors to regulate cAMP production and, consequently, sperm function. Although the experiments discussed here have been carried out in vitro, it is plausible that these molecules might be able to affect spermatozoa in vivo as well. If so, they could accelerate capacitation and then hold the spermatozoa in a state of readiness to fertilize by inhibiting spontaneous acrosome loss, thereby possibly increasing the chances of conception. Way et al. (2001) reported detecting low-to-moderate concentrations of noradrenaline (100–220 nmol/l) in the oviductal secretions of cows around the time of ovulation, concentrations well within the range that elicit responses in mouse and human spermatozoa. It is therefore possible that reproductive tract secretions in human females also have noradrenaline that could act on spermatozoa. In addition, many individuals use amphetamine-related compounds for either medical or social reasons, and these could also have a positive effect on sperm function, especially if they have a relatively long half-life, once ingested. For example, 20 h or more after individuals had chewed khat leaves for 1 h, cathine and norephedrine were still present in their blood at concentrations we have shown to act on spermatozoa (Toennes et al., 2003). Given our new evidence that such compounds can act directly on spermatozoa via adrenergic receptors, it would be prudent for individuals taking amphetamine-related compounds to be aware that there might be unexpected side effects that could include an increased chance of conception.
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We would like to thank Kate Kirwan, Manager of the Photography & Digital Imaging Unit, School of Biomedical and Health Sciences, King's College London, for her invaluable assistance in preparing Figures 6 and 7.
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Submitted on August 18, 2006; resubmitted on October 24, 2006; accepted on October 26, 2006.
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