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Reproduction (2004) 127 601-612
DOI: 10.1530/rep.1.00062
Copyright © 2004 Society for Reproduction and Fertility
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RESEARCH

Angiotensin II stimulates cAMP production and protein tyrosine phosphorylation in mouse spermatozoa

Samra Mededovic and Lynn R Fraser

Centre for Reproduction, Endocrinology and Diabetes, School of Biomedical Sciences, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK

Correspondence should be addressed to L R Fraser; Email: lynn.fraser{at}kcl.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Angiotensin II (AII), found in seminal plasma, has been shown to stimulate capacitation in uncapacitated mammalian spermatozoa. The present study investigated the location of AII receptors on spermatozoa and AII’s mechanism of action. AT1 type receptors for AII are present on the acrosomal cap region and along the whole of the flagellum of both mouse and human spermatozoa. Because combinations of low concentrations of AII and either calcitonin or fertilization-promoting peptide (FPP), both known to regulate the adenylyl cyclase (AC)/cAMP signal transduction pathway, elicited a significant response, this study investigated the hypothesis that these peptides act on the same pathway. AII was shown to significantly stimulate cAMP production in both uncapacitated and capacitated mouse spermatozoa and this was associated with increases in protein tyrosine phosphorylation. Using an anti-phosphotyrosine antibody to visualize the location of tyrosine phosphoproteins within individual cells, AII significantly stimulated phosphorylation within 20 min in both the head, especially in the acrosomal cap region, and the flagellum, especially in the principal piece, of uncapacitated mouse spermatozoa; combined AII + FPP was stimulatory within 5 min. In addition, Western blotting revealed that AII stimulation increased phosphorylation in a number of tyrosine phosphoproteins in both uncapacitated and capacitated mouse spermatozoa, with some being altered only in the latter category of cells. These results support the hypothesis that AII stimulates AC/cAMP in mammalian spermatozoa.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Successful fertilization is the result of complex molecular events that enable mature spermatozoa to recognize, bind to and fuse with the oocyte. To achieve this success, spermatozoa must undergo a number of maturational changes that occur during transit through the epididymis. In addition, they must undergo further post-release maturation, termed ‘capacitation’ (Austin 1952), either in the female reproductive tract or in appropriate culture media in vitro. The biochemical and physiological changes that underpin capacitation are still poorly understood, but upon completion of these changes, spermatozoa possess the capacity to fertilize an oocyte (Yanagimachi 1994, de Lamirande et al. 1997). During the past few years, investigations have revealed that several small molecules present in seminal plasma can bind to specific receptors on mammalian spermatozoa and elicit biologically important responses; these cause the spermatozoa to ‘switch on’ more quickly, resulting in accelerated capacitation. These molecules include fertilization-promoting peptide (FPP), adenosine and calcitonin, all of which have been shown to stimulate capacitation in uncapacitated spermatozoa and then inhibit spontaneous acrosome reactions in capacitated cells (Fraser & Adeoya-Osiguwa 2001).

Angiotensin II (AII) is an octapeptide (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) widely known for its roles in regulating cardiovascular and electrolyte homeostasis (see review by Vinson et al. 1997). However, AII is also found in seminal plasma at concentrations higher than in blood plasma (O’Mahony et al. 2000) and there is evidence that AII receptors of subtype 1 (AT1) are present on mammalian spermatozoa (Vinson et al. 1995, Gur et al. 1998, Wennemuth et al. 1999). More recently, Fraser et al.(2001) demonstrated that AII, like FPP, adenosine and calcitonin, stimulated capacitation and demonstrable fertilization in vitro in uncapacitated mouse spermatozoa but, unlike those molecules, AII had no inhibitory effects on capacitated cells. When low, ineffective concentrations of AII were used in combination with low concentrations of either FPP or calcitonin or with both, a significant response was obtained in uncapacitated cells, suggesting that the peptides were acting on the same signal transduction pathway. Since considerable evidence indicates that FPP, adenosine and calcitonin all regulate adenylyl cyclase (AC)/cAMP, initially stimulating cAMP production in uncapacitated cells and then inhibiting cAMP in capacitated cells (Adeoya-Osiguwa et al. 1998, Fraser & Adeoya-Osiguwa 1999, Adeoya-Osiguwa & Fraser 2002, 2003), it was hypothesized that AII also acts via that pathway.

The present study was undertaken to (i) localize AII receptors on mature mouse and human spermatozoa and then (ii) investigate AII’s mechanism of action. Although two main subtypes of AII receptors, AT1 and AT2, have been identified, the AT2 receptors are found primarily in fetal tissues and in pathophysiological conditions (Nishimura 2001); experimental evidence from many adult tissues suggests that AT1 receptors predominate (Sayeski et al. 1998, Nishimura 2001) and there is some evidence for AT1 receptors in spermatozoa. However, earlier studies produced conflicting results, with Vinson et al.(1995) reporting AT1 receptors to be located on the flagella of mature rat and human spermatozoa and Wennemuth et al.(1999) reporting that these receptors were located on both the acrosomal region of the head and the principal piece of mature mouse spermatozoa. Given that AII would need to cause changes in both the head and the flagellar compartments in order to accelerate capacitation and demonstrable fertilizing ability, we hypothesized that AT1 receptors would be present in both regions. Subsequent experiments were designed to determine whether AII had any effect on cAMP production and protein tyrosine phosphorylation in both uncapacitated and capacitated mouse spermatozoa, the hypothesis being that AII was likely to stimulate both cAMP and phosphorylation in cells at both functional stages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
All animals in this study were minatained in Home Office-approved facilities.

Media and reagents
The standard medium used in these experiments was a modified Tyrode’s medium (Fraser 1993) containing 4 mg/ml BSA. Unless otherwise specified, all reagents were obtained from Sigma (Poole, Dorset, UK). Concentrated stock solutions of human AII were prepared in medium, divided into aliquots, frozen and kept at –20 °C. FPP (pGlu-Glu-ProNH2) solutions were prepared as described previously (Green et al. 1994), lyophilized and stored at –20 °C. For use, samples were reconstituted in BSA-free medium, aliquoted and frozen; these were kept for a maximum of 1 month. In all experiments, the final working stock of peptide was 50 x final desired concentration. Stock solutions of CGS 21680 (a specific stimulatory A2A adenosine receptor agonist), N6-cyclopentyladenosine (CPA), a specific inhibitory A1 adenosine receptor agonist) and the cyclic nucleotide phosphodiesterase inhibitor Ro-20-1724 were prepared daily in DMSO. Each was diluted further in complete medium such that the final DMSO concentration was less than 0.1%, a concentration shown to have no adverse effects on sperm function (Fraser 1990). For immunolocalization of receptors, specific antibodies against AT1 receptors were obtained from Santa Cruz Biotechnology (Autogen Bioclear, Calne, Wiltshire, UK): AT1 (N-10) and AT1 (306). When available, appropriate blocking peptides were also purchased. For immunolocalization of tyrosine phosphoproteins, antibody 4G10 was obtained from Upstate Biotechnology (Buckingham, UK).

Sperm suspension preparation
For functional assays using either uncapacitated or capacitated mouse sperm suspensions, cauda epididymides from mature male TO mice (Harlan UK, Bicester, Oxon, UK) were removed and the contents were extruded into a 30 mm sterile culture dish (Nunc, Roskilde, Denmark) containing modified Tyrode’s medium (+CaCl2, +BSA; 0.8 ml medium per pair of caudae). Suspensions were maintained on a warming tray (~37 °C) and allowed to disperse for 5 min. In experiments using uncapacitated spermatozoa, suspensions were used immediately while in those using capacitated cells, suspensions were incubated for 90 min under autoclaved liquid paraffin (Boots, Nottingham, UK) at 37 °C in an atmosphere of 5% CO2: 5% O2: 90% N2. Just before use, all suspensions were filtered through short columns (Pasteur pipettes plugged with a small amount of glass wool) of Sephadex G-25 (medium grade; Amersham Biosciences) pre-equilibrated with standard medium to remove non-motile cells. When mouse spermatozoa are preincubated in this manner, we have shown many times that they are capacitated as evidenced by chlortetracycline analysis (e.g. Fraser et al. 2001), in vitro fertilization (e.g. Fraser 1993) and tyrosine phosphorylation analysis (Adeoya-Osiguwa & Fraser 2000).

For immunolocalization of AT1 receptors, uncapacitated mouse sperm suspensions were prepared in medium with no added CaCl2 and with 4 mg/ml polyvinyl alcohol (cold water soluble) in place of BSA; this medium maintains sperm viability but does not support capacitation. After dispersal, suspensions were filtered as described above to remove non-motile cells and then used. For immunolocalization in capacitated mouse spermatozoa, suspensions were prepared in complete medium (+CaCl2, +BSA), incubated as described above for 90 min, filtered and then used. Human semen samples were provided by healthy adult normal donors (with approval from the King’s College Research Ethics Committee). Semen was layered onto a discontinuous 95%/70%/50% Percoll (Amersham) gradient prepared in Earle’s medium (DasGupta et al. 1993) and centrifuged at 600 g for 5 min; the pellet containing mainly motile uncapacitated spermatozoa was removed, washed once in fresh medium, resuspended at ~5 x 106 cells/ml and used as described below.

Immunolocalization of AT1 receptor
As soon as mouse and human sperm suspensions had been prepared, an equal volume of 4% w/v sucrose was added to stabilize cells. Pilot studies were undertaken to determine the best way to prepare cells, e.g. unpermeabilized vs permeabilized, which method of permeabilization, length and temperature of incubation with antibody, etc. Of the three different methods assessed for permeabilization (liquid N2, dry ice/acetone and 0.2% v/v Triton X-100), the best and most consistent staining was obtained with liquid N2 permeabilization. Unpermeabilized cells were fixed with 2.5% paraformaldehyde in PBS, with gentle agitation for 30 min at room temperature. Permeabilized cells were prepared by freezing in liquid N2, thawing at room temperature and fixing with paraformaldehyde as for unpermeabilized cells. Spermatozoa were centrifuged at 600 g for 6 min; pelleted cells were resuspended in the same volume of PBS and applied to microscope slides coated with 0.1% w/v poly-L-lysine in distilled water. After 10 min, slides were washed in PBS and the antibody was applied; both AT1 (306) and AT1 (N-10) were used at 1:25, diluted in PBS containing 3% BSA. Slides were incubated overnight at room temperature (conditions found to give optimal results), washed in PBS and biotinylated anti-rabbit IgG was added (H + L; Vector Laboratories, Peterborough, UK); the latter was diluted 1:100 with 10 mM phosphate buffer/0.15 M NaCl, pH 7.8. After 1 h at room temperature in a humid chamber, slides were washed in PBS and then FITC-Avidin D (Vector), diluted 1:250 with 0.1 M NaHCO3/0.15 M NaCl, pH 8.5 buffer, was added; slides were incubated for 30 min at room temperature in a humid chamber (in the dark). Slides were washed well in PBS and then a drop of 0.22 M 1,4-diazabicyclo[2.2.2]octane in glycerol:PBS (9:1), an anti-fade agent, was added, followed by a cover slip; excess liquid was removed by pressing down with a tissue and the preparation was sealed with colourless nail varnish. Slides were stored, wrapped in foil, at 4 °C.

Cells were assessed using an Olympus BX40 microscope (Olympus Optical Co. (UK), Southall, Middlesex, UK) equipped with phase contrast and BX-FLA epifluorescence optics using the wide blue excitation cube (U-MWB). The excitation beam was passed through a 450–480 nm band pass filter and fluorescence emission was observed through a DM500 dichroic mirror. Receptor localizations shown and discussed below are typical of results from more than five replicate experiments each for uncapacitated and capacitated suspensions.

cAMP measurement
The amount of cAMP produced in live, intact cells was determined using a non-radioactive enzyme immunoassay kit from Amersham. Assays were performed as described in the instructions provided with the kit.

Immunolocalization of tyrosine phosphoproteins
The immunolocalization techniques used in this series are based on those described by Urner et al.(2001). Sperm suspensions were prepared and filtered either immediately (uncapacitated) or after 90 min incubation under liquid paraffin at 37 °C in 5% CO2:5% O2:90% N2 (capacitated). Sodium orthovanadate (100 µM final concentration) was added to suspensions prior to treatment with individual or combined peptides in order to inhibit phosphotyrosine phosphatases (Swarup et al. 1982). At the desired time point, reactions were stopped by adding aliquots of the sperm suspension to a tube containing reagents to give final concentrations of 4 mM EDTA, 0.2 µg/ml leupeptin and 400 µg/ml trypsin inhibitor (type I-S, soybean) plus 2% w/v sucrose. Cells were fixed with 2.5% paraformaldehyde and centrifuged at 600 g for 6 min; pelleted cells were resuspended in the same volume of PBS and applied to 0.1% poly-L-lysine coated slides. Spermatozoa were then permeabilized by addition of 0.2% Triton X-100 in PBS for 10 min. Slides were washed in PBS, incubated in 10% horse serum (blocking agent) for 1 h at room temperature and washed again. Anti-phosphotyrosine antibody 4G10, diluted 1:100 in PBS/3% BSA, was added and slides were incubated overnight at room temperature. After washing, biotinylated anti-mouse IgG (H + L; Vector; diluted 1:100 with 10 mM phosphate buffer/0.15 M NaCl, pH 7.8) was added for 1 h at room temperature in a humid chamber. After washing, FITC-Avidin D (Vector), diluted 1:250 with 0.1 M NaHCO3/0.15 M NaCl, pH 8.5 buffer, was added and slides were incubated for 30 min at room temperature in a humid chamber (in the dark). Slides were washed well in PBS and semi-permanent preparations were made as described above.

Electrophoresis, Western blotting and detection of tyrosine phosphoproteins
In all experiments, sodium orthovanadate was added to filtered suspensions to give a final concentration of 100 µM. Suspensions were treated experimentally for various times and then 200–400 µl of each sample was transferred to plastic conical microcentrifuge tubes containing reagents to give final concentrations of 4 mM EDTA, 0.2 µg/ml leupeptin and 400 µg/ml trypsin inhibitor, then immediately frozen in liquid N2. Cells were thawed at room temperature, pelleted by centrifugation at 11 000 g at 4 °C for 5 min to remove BSA and then resuspended in PBS containing 4 mM EDTA, 0.2 µg/ml leupeptin, 400 µg/ml trypsin inhibitor and 100 µM sodium orthovanadate to give a final sperm concentration of ~3.5 x 107 cells/ml for uncapacitated and ~2 x 107 cells/ml for capacitated suspensions. Cells were solubilized in electrophoresis loading buffer (6% SDS, 13.5% glycerol, 150 mM dithiothreitol, 100 mM Tris–HCl, pH 6.8, and 0.03% bromphenol blue), then denatured in a boiling water bath for 7 min. Proteins were resolved on 8–10% linear gradient Tris–glycine acrylamide gels with a 4% stacking gel; gels were prepared using Protogel reagents (bis/acrylamide, buffer and stacking buffer) and protocols (National Diagnostics, Hull, UK) and were routinely run at 200 V for ~45 min. Proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes (Hybond P; Amersham) at 0.8 mA/cm2 for 1 h.

PVDF membranes were blocked with 1% BSA in Tris-buffered saline (10 mM Tris–HCl/100 mM NaCl, pH 7.5) + 0.1% Tween (TBST) for 1 h at room temperature with gentle agitation. Membranes were then washed twice with fresh TBST to remove blocking agent and incubated with gentle agitation for 2 h at room temperature with anti-phosphotyrosine–horseradish peroxidase conjugate (PY20-HRP, Prod. No. RPN2221; Amersham) diluted in TBST (1:1000 for uncapacitated and 1:2000 for capacitated cells). Membranes were washed three times in TBST to remove excess antibody and then developed using the ECL plus kit (Amersham). Specifically bound antibody was detected by exposing the PVDF membrane to X-ray film (Fuji Film), with Fuji intensifying screens. Films were developed and photo quality prints were obtained using PhotoShop (Adobe PhotoShop 4.0 LE).

It should be noted that fewer cells and a more dilute antibody solution were used for assessment of capacitated suspensions because the level of phosphorylation is much higher than in uncapacitated suspensions (e.g. Adeoya-Osiguwa & Fraser 2000).

Statistical analysis
Analysis of data from tyrosine phosphoproteins localization used Cochran’s modification of the {chi}2 test (Snedecor & Cochran 1980). Analysis of data from cAMP determinations used a paired t-test (Sigma Stats; Jandell Scientific International, Chicago, IL, USA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Series I: localization of AT1 receptors
Of the two antibodies used, the AT1 (306) antibody, directed against a sequence in the C-terminal region of the receptor, gave stronger and more consistent fluorescence than did the AT1 (N-10) antibody, directed against a sequence in the N-terminal region. As expected, a signal was obtained only when permeabilized cells were used with AT1 (306) since the epitope is intracellular, but even AT1 (N-10) worked better on permeabilized than non-permeabilized cells. We have made similar observations with antibodies directed against both the N-terminal and the C-terminal regions of other membrane-spanning receptors. This is presumably because the N-terminal epitope chosen for the antigen is said by the supplier to be ‘near’ the end, rather than at the very end, of the protein and so may be at least partly embedded in the plasma membrane; thus permeabilization would increase antibody access to the epitope. Appropriate control treatments were included. When the AT1 (N-10) antibody was preincubated with the relevant blocking peptide, no signal was observed. A blocking peptide was not available for AT1 (306), but omission of the primary antibody abolished the signal.

Both uncapacitated and capacitated mouse spermatozoa were evaluated to determine whether there were any detectable capacitation-dependent alterations such as those reported for adenosine receptors by Adeoya-Osiguwa & Fraser (2002), but no differences were noted. With both antibodies, staining was observed in the acrosomal cap and along the whole of the flagellum in uncapacitated mouse (Fig. 1aGo), capacitated mouse (Fig. 1bGo) and uncapacitated human (Fig. 1cGo) spermatozoa; in addition, some staining in the posterior part of the postacrosomal region (just above the neck) was seen reasonably frequently.



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  		Figure 1 Immunofluorescent localization of AT1 receptors on mouse and human spermatozoa using antibody AT1 (306). Staining in the acrosomal cap region, the neck and the flagellum can be seen in (a) uncapacitated mouse, (b) capacitated mouse and (c) uncapacitated human spermatozoa. The bar represents 10 µm for mouse and 5 µm for human gametes.

 
Series II: AII stimulates cAMP production in both uncapacitated and capacitated mouse spermatozoa
In experiments evaluating effects of AII on cAMP production, appropriate positive controls were included. With uncapacitated suspensions, CGS 21680 was used; this is a specific agonist that binds to stimulatory A2A adenosine receptors and stimulates cAMP production in uncapacitated spermatozoa (Fraser & Adeoya-Osiguwa 1999). With capacitated suspensions, CPA was used; this is a specific agonist that binds to inhibitory A1 adenosine receptors and inhibits cAMP production in capacitated spermatozoa (Adeoya-Osiguwa & Fraser 2002).

For investigations on uncapacitated spermatozoa, sperm suspensions were prepared in complete medium, allowed to disperse and then filtered (n = 6). The filtered suspension was divided into three and treated as follows: (i) untreated control; (ii) 10 nM AII; and (iii) 500 nM CGS 21680. Just prior to these treatments, the phosphodiesterase inhibitor Ro-20-1724 (5 µM final concentration) was added to each sample. Samples were incubated for 2 min at 37 °C; to stop the reaction, 200 µl of each suspension were transferred into tubes containing reagents to give final concentrations of 4 mM EDTA+0.2 µg/ml leupeptin + 400 µg/ml trypsin inhibitor and then frozen in liquid N2. After thawing at room temperature, cAMP was extracted with ethanol and determinations were carried out as described in the kit instruction booklet. AII significantly stimulated cAMP production when compared with control samples (Fig. 2aGo), as did CGS 21680.



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  		Figure 2 AII stimulates cAMP production in both uncapacitated and capacitated mouse spermatozoa. (a) In uncapacitated suspensions (n = 6), both 10 nM AII and 500 µM CGS 21680, an agonist acting on stimulatory A2A adenosine receptors, significantly stimulated cAMP, compared with untreated controls (Control); samples were assessed at 2 min. (b) In capacitated suspensions (n = 6), 10 nM AII significantly stimulated cAMP while 5 nM CPA, an agonist acting on inhibitory A1 adenosine receptors, significantly inhibited cAMP, compared with untreated controls; samples were assessed at 5 min; data are presented as means±S.E. **P < 0.025 compared with relevant control suspensions.

 
For investigations on capacitated spermatozoa, suspensions were prepared in 30 mm sterile culture dishes, covered with liquid paraffin and incubated for 90 min. They were then filtered, divided into three and treated with (i) nothing; (ii) 10 nM AII; or (iii) 5 nM CPA; Ro-20-1724 was added before test reagents. Samples were incubated for 5 min, the reaction was stopped and cAMP was extracted as above (n = 6). Again AII significantly stimulated cAMP production while CPA significantly inhibited it, compared with the untreated control suspensions (Fig. 2bGo).

Series III: immunolocalization of tyrosine phosphoproteins in AII-treated uncapacitated spermatozoa
Sperm suspensions were prepared in complete medium, allowed to disperse for 5 min, filtered and sodium orthovanadate (100 µM final concentration) was added to inhibit phosphatase activity. In the first series (n = 6), suspensions were divided into aliquots and treated with: (i) nothing (control); (ii) 10 nM AII; (iii) 100 nM FPP (positive control); or (iv) 10 nM AII + 100 nM FPP. Suspensions were gassed with 5% CO2, 5% O2, 90% N2 and then incubated at 37 °C for 20 min. In the second series (n = 5), suspensions were treated with (i) nothing (control) or (ii) 10 nM AII + 100 nM FPP and incubated for a total of 20 min, with samples being removed for assessment with anti-phosphotyrosine antibody 4G10 at 5, 10, 15 and 20 min. In both series, the presence of fluorescence within different regions of individual cells (acrosomal cap, equatorial segment, postacrosomal region, midpiece and principal piece) and the intensity of fluorescence (absent, weak or strong) were evaluated. In the second series, cells were also scored as having staining in: neither head nor flagellum, head only, flagellum only or head + flagellum. In all experiments, 100 cells were evaluated in each treatment group. A typical staining pattern is shown in Fig. 3aGo.



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  		Figure 3 Immunolocalization of tyrosine phosphoproteins in uncapacitated and capacitated mouse spermatozoa. Typical patterns of fluorescence seen after permeabilization and incubation with anti-phosphotyrosine antibody 4G10 followed by a biotinylated secondary antibody and FITC-Avidin D are shown in (a) for uncapacitated cells with strong staining in both the acrosomal cap and the principal piece and (c) for capacitated cells with strong staining in the flagellum; corresponding phase contrast images of the same cells (b, d) are on the right. The bar represents 10 µm.

 
In the first series, it could be seen that AII and FPP, used individually, significantly stimulated tyrosine phosphorylation as evidenced by stronger fluorescence, compared with the control (Fig. 4Go). Of the four compartments assessed, only the acrosomal cap region and the principal piece showed any change. No differences were detected in either the equatorial segment or the midpiece of untreated and treated cells and, indeed, the majority of cells had very little or no staining in these regions; no staining was seen in the postacrosomal region. Surprisingly, the combined AII + FPP had no detected effect. However, since both peptides stimulate cAMP production in uncapacitated cells, the response to combined peptide would probably occur more quickly and so a second set of experiments was carried out with sampling at 5 min intervals from 5–20 min. Results revealed that the combination treatment did elicit responses more quickly; by 5 min (Fig. 5Go) there was significantly more fluorescence in the acrosomal cap region and principal piece of the peptide-treated cells and there was also a slight stimulation in the midpiece region. This response was clearly tapering off by 10 min and by 15 min there were no significant differences between control and treated suspensions (data not shown). When cells were analysed by evaluating the presence of fluorescence in the head, flagellum, both or neither, the combined peptide treatment for 5 min significantly increased the number of cells with staining in both compartments, with concomitant decreases in those with no staining and staining in the head only (Fig. 6Go). The response, although still significantly different, was clearly lessening by 10 min and had disappeared by 15 min (data not shown).



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  		Figure 4 Relative intensity of fluorescence indicating presence of tyrosine phosphoproteins in uncapacitated mouse spermatozoa. Suspensions (n = 6) were incubated for 20 min following the addition of (i) nothing (Control); (ii) 100 nM FPP; (iii) 10 nM AII; or (iv) 10 nM AII + 100 nM FPP and prepared for visualization of tyrosine phosphoproteins. Cells were assessed as having no staining (left bar in each group of three), weak staining (middle bar) or intense staining (right bar) in four different regions: (a) acrosomal cap; (b) equatorial segment; (c) midpiece; (d) principal piece. Data are presented as means±S.E. *P < 0.05, **P < 0.025 compared with untreated controls.

 


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  		Figure 5 Combined AII and FPP elicit more rapid changes in tyrosine phosphoproteins. Uncapacitated suspensions (n = 5) were incubated for 5 min following the addition of (i) nothing (Control) or (ii) 10 nM AII + 100 nM FPP, then assessed for phosphoproteins. Cells were assessed as having no staining (left bar in each group of three), weak staining (middle bar) or intense staining (right bar) in four different regions: (a) acrosomal cap; (b) equatorial segment; (c) midpiece; (d) principal piece. Data are presented as means±S.E. *P < 0.05, **P < 0.025, ***P < 0.01 compared with untreated controls.

 


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  		Figure 6 Combined AII and FPP stimulate phosphorylation in both the head and the flagellum. Uncapacitated suspensions (n = 5) were incubated for 5 min following the addition of (i) nothing (Control) or (ii) 10 nM AII + 100 nM FPP, then assessed. Cells were assessed as having no staining (left bar in each group), staining in head only (second bar), staining in flagellum only (third bar), or staining in both head and flagellum (right bar). Data are presented as means±S.E. **P < 0.025, ****P < 0.001 compared with untreated controls.

 
Series IV: immunolocalization of tyrosine phosphoproteins in AII-treated capacitated spermatozoa
For capacitated spermatozoa, dispersed suspensions were covered with liquid paraffin, incubated for 90 min, filtered and treated; as previously, 100 µM sodium ortho-vanadate (final concentration) was added prior to treatment. In these experiments (n = 4), suspensions were divided into two and treated with (i) nothing (control) or (ii) 10 nM AII for a total of 20 min, with samples being taken at both 10 and 20 min. As in Series III, cells were analysed both in terms of staining in specific regions (acrosomal cap, equatorial segment, midpiece, principal piece) and in head/flagellum/both/neither. Analysis of regions at both time points gave very similar results for both AII-treated and untreated suspensions (Fig. 7Go) and revealed that most cells had strong staining in both the midpiece and principal piece (Fig. 3cGo); in the minority of cells with staining in the head, this was primarily in the equatorial segment. Similarly, analysis of head and flagellar compartments at both time points showed that there was no detected response to AII; the majority of cells in both groups had staining only in the flagellum and about a third of the cells had staining in both compartments (Fig. 8Go).



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  		Figure 7 Relative abundance of tyrosine phosphoproteins in capacitated mouse spermatozoa. Suspensions (n = 4) were incubated for 20 min following the addition of (i) nothing (Control) or (ii) 10 nM AII and prepared for visualization of tyrosine phosphoproteins. Cells were assessed for no staining (left bar in each group), weak staining (middle bar) or intense staining (right bar) in four different regions: (a) acrosomal cap; (b) equatorial segment; (c) midpiece; (d) principal piece. Data are presented as means±S.E. No significant differences could be detected between untreated and treated suspensions.

 


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  		Figure 8 Tyrosine protein phosphorylation is most prominent in flagella of capacitated mouse spermatozoa. Capacitated suspensions (n = 5) were incubated for 20 min following the addition of (i) nothing (Control) or (ii) 10 nM AII, then assessed. Cells were assessed as having no staining (left bar of each group), staining in head only (second bar), staining in flagellum only (third bar), or staining in both head and flagellum (right bar); data are presented as means±S.E. No significant differences could be detected between untreated and treated suspensions.

 
Series V: AII stimulates tyrosine phosphorylation of a number of phosphoproteins in both uncapacitated and capacitated spermatozoa
Filtered uncapacitated suspensions were divided into four aliquots and then treated with: (i) nothing (control); (ii) 100 nM FPP; (iii) 10 nM AII; or (iv) 100 nM FPP + 10 nM AII. Suspensions were incubated at 37 °C for 10–20 min and then evaluated using gel electrophoresis and Western blotting. Within 10 min of peptide addition to suspensions, phosphorylation was clearly stimulated, especially with combined peptide treatment, when compared with the untreated controls (Fig. 9Go). In particular, phosphoproteins of ~35, 42, 56, 66, 75, 82, 95 and 116 kDa were either absent or less heavily phosphorylated in the untreated controls, compared with peptide-treated suspensions.



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  		Figure 9 Western blots of tyrosine phosphoproteins present in uncapacitated mouse spermatozoa. Uncapacitated suspensions were treated with (i) nothing (Con); (ii) 100 nM FPP; (iii) 10 nM AII; or (iv) 10 nM AII + 100 nM FPP; samples were taken at 10 min and assessed. The position of standards is shown to the left of the figure. Enhanced phosphorylation of several proteins (denoted by a dot) was detected in suspensions treated with individual and combined peptides.

 
Filtered capacitated suspensions were divided into aliquots and treated as above. Within 10 min of peptide addition, it was possible to see differences in the degree of phosphorylation in the peptide-treated samples and the untreated controls. Although both FPP and AII had stimulatory effects on uncapacitated cells, in capacitated suspensions FPP inhibited phosphorylation while AII still stimulated it (Fig. 10aGo). In particular, there was reduced or absent phosphorylation in phosphoproteins of ~42, 50, 56, 66, 70, 75, 82, 95 and 116 kDa in FPP-treated suspensions, but enhanced phosphorylation of these phosphoproteins in AII-treated suspensions. The phosphoproteins of 50 and 70 kDa were not among those stimulated in uncapacitated suspensions. When suspensions were treated with a combination of AII + FPP, the inhibitory effects of FPP were able to override any responses to AII (Fig. 10bGo). This is consistent with earlier studies demonstrating that although AII did not inhibit acrosome loss in capacitated spermatozoa, acrosome loss was significantly inhibited in suspensions treated with AII + FPP (Fraser et al. 2001).



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  		Figure 10 Western blots of tyrosine phosphoproteins present in capacitated mouse spermatozoa. In (a), capacitated suspensions were treated with (i) nothing (Con) or (ii) 100 nM FPP or 10 nM AII for 10 min and assessed. In (b), suspensions were treated with (i) nothing (Con) or (ii) 10 nM AII + 100 nM FPP for 5 min and assessed. Although AII on its own stimulated tyrosine phosphorylation of several proteins (denoted by a dot), this response was inhibited when AII was used in combination with FPP.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
AII has been shown to significantly accelerate capacitation and demonstrable fertilizing ability in mouse spermatozoa (Fraser et al. 2001). Since fertilization requires changes in both the head, to prepare for the acrosome reaction, and the flagellum, to promote hyperactivated motility, it was hypothesized that the AII receptor AT1 would be located in both compartments of the cell. Indeed, results indicated that AT1 is located in the acrosomal cap region and along the entire flagellum in both mouse and human uncapacitated spermatozoa (Fig. 1Go); no obvious differences were noted when distributions on uncapacitated and capacitated mouse gametes were compared. These locations are similar to those described by Wennemuth et al.(1999) for mouse spermatozoa, except that labelling was present all along the flagellum and not just in the principal piece, but dissimilar to those reported by Vinson et al.(1995) for human and rat gametes, where localization using an in-house monoclonal antibody was observed only on the flagellum. However, two different commercially available antibodies, each directed against a different region of the receptor, and a variety of preparation methods were evaluated in the present study; staining was consistently observed in both compartments of human, as well as mouse, spermatozoa. Given that human spermatozoa respond physiologically to AII in a manner analogous to mouse gametes (Fraser & Osiguwa 2004) and that those responses involve changes in both head and flagellum, the present results would appear to be biologically plausible.

The demonstration that combinations of low concentrations of AII plus low FPP or calcitonin significantly stimulated capacitation in uncapacitated mouse (Fraser et al. 2001) and human (Fraser & Osiguwa 2004) spermatozoa suggested that all three peptides affect the same signalling pathway, known to be AC/cAMP for FPP and calcitonin. Current evidence indicates that FPP receptors elicit these responses by interacting with stimulatory and inhibitory adenosine receptors (Fraser & Adeoya-Osiguwa 1999). To test the hypothesis that AII affects AC/cAMP, uncapacitated mouse sperm suspensions were treated with CGS 21680, a specific agonist acting at stimulatory A2A adenosine receptors and shown previously to stimulate AC/cAMP (Fraser & Adeoya-Osiguwa 1999), and with AII. Capacitated suspensions were treated with CPA, a specific agonist acting at inhibitory A1 adenosine receptors and shown to inhibit AC/cAMP in similar suspensions (Adeoya-Osiguwa & Fraser 2002), and with AII. As expected, CGS stimulated cAMP and CPA inhibited cAMP, but AII significantly stimulated cAMP production in both uncapacitated and capacitated suspensions (Fig. 2Go). These results are consistent with earlier observations that adenosine initially stimulates capacitation and then inhibits spontaneous acrosome reactions (Green et al. 1996), while AII initially stimulates capacitation but subsequently does not inhibit spontaneous acrosome loss (Fraser et al. 2001). Several papers published recently (bovine, Gur et al. (1998); human, Köhn et al.(1998); equine, Sabeur et al.(2000)) have reported that AII stimulates the acrosome reaction, but the experimental protocols involved either long or complicated incubations in the presence of AII or preincubation in a cAMP analogue followed by AII. However, the demonstration that AII stimulates cAMP throughout capacitation would be consistent with accumulation of cAMP until a threshold value has been reached that would then stimulate spontaneous acrosome reactions. This could explain the results in those other studies.

The present results led to the hypothesis that significant increases in cAMP obtained with AII would stimulate protein kinase A, followed by tyrosine kinase, and so result in increased protein tyrosine phosphorylation (e.g. Visconti et al. 1995, Adeoya-Osiguwa & Fraser 2000). This was investigated in both uncapacitated and capacitated mouse spermatozoa using two different approaches. First, an anti-phosphotyrosine antibody was used to visualize location and general abundance of tyrosine phosphoproteins in different regions of mouse sperm heads and flagella; secondly, the more conventional approach of PAGE/Western blotting was employed to identify specific tyrosine phosphoprotein bands showing differences in the degree of phosphorylation. In these experiments, AII and FPP were used individually and in combination; FPP was a useful comparator since it had already been shown initially to stimulate and then to inhibit tyrosine phosphorylation (Adeoya-Osiguwa & Fraser 2000). Used individually, both AII and FPP significantly stimulated tyrosine phosphorylation in the heads and flagella of uncapacitated cells within 20 min as evidenced by greater fluorescence (Fig. 4Go); interestingly, though, within each compartment the changes were only seen in one region. In the head, there was clearly an increase in fluorescence in the acrosomal cap region in the treated suspensions, compared with untreated controls, but there was generally little or no staining in the equatorial segment and essentially no staining in the postacrosomal regions in either control or experimental samples. In the flagellum, the marked changes occurred in the principal piece while there was relatively little fluorescence in the midpiece, irrespective of treatment. Physiologically, these changes in phosphorylation are presumably involved in preparing spermatozoa eventually to undergo an acrosome reaction upon contacting an oocyte and to express hyperactivated motility.

Given those results with individual peptides, it was puzzling that treatment for 20 min with AII + FPP resulted in no significant differences, compared with untreated controls. Upon reflection, however, it seemed likely that the combined treatment would elicit responses more quickly and so suspensions treated with the combination were sampled at 5 min intervals from 5–20 min. Within 5 min (Fig. 5Go), significant stimulation of phosphorylation could be detected in both the acrosomal cap and the principal piece, the degree of stimulation being similar in magnitude to that obtained after 20 min with individual peptides (Fig. 4Go). When cells were evaluated for staining in head, flagellum, both or neither, by 5 min a marked increase in the proportion of spermatozoa showing fluorescence in both head and flagellum was observed, compared with controls (Fig. 6Go); as with the other assessment approach, stimulation had lessened by 10 min and then had disappeared by 15 min. Thus the combination of AII + FPP does elicit a significant response but it occurs much more quickly than in suspensions treated with individual peptides.

When the same technique was used on capacitated spermatozoa, there were no detected differences between AII-treated and untreated suspensions (Figs 7Go and 8Go). The majority of fluorescence was seen in the flagellum, both the midpiece and the principal piece, and about a third of cells also had staining in the head, mainly in the equatorial segment. In other studies using a similar technical approach to evaluate tyrosine phosphorylation in capacitated human (Bajpai & Doncel 2003) and dog (Petrunkina et al. 2003) spermatozoa, the majority of positive cells had fluorescence primarily in the flagellum, with some staining also in the equatorial segment, as in the present study. This suggests that these events are a general occurrence in many mammalian spermatozoa (see also Urner & Sakkas 2003) and could well be associated with expression of hyperactivated motility. Although AII significantly stimulated cAMP in capacitated cells, there were no detected differences in the amount of fluorescence between the treated and untreated cells. However, the general level of tyrosine phosphorylation is much greater in capacitated than in uncapacitated control spermatozoa (Adeoya-Osiguwa & Fraser 2000) and it seems likely that this experimental approach is not sufficiently sensitive to pick up possible differences in capacitated cells.

PAGE/Western blotting is more sensitive and allows identification of specific tyrosine phosphoproteins with altered phosphorylation states in cells treated with various peptides. In uncapacitated cells, phosphoproteins present in suspensions incubated with AII, FPP and AII + FPP were compared with those in untreated controls. Individual and combined peptide treatment significantly stimulated the phosphorylation of a number of proteins (Fig. 9Go); those showing the greatest stimulation were 35, 42, 56, 66, 75, 82, 95 and 116 kDa; proteins of these sizes were also noted in the study of Adeoya-Osiguwa & Fraser (2000). For many of these proteins, there was even more intense staining in suspensions incubated with the combined peptides than with individual peptides. In capacitated cells, FPP inhibited phosphorylation while AII continued to stimulate it (Fig. 10aGo); however, when the combination of AII + FPP was used, phosphorylation was inhibited, consistent with evidence that FPP exerts its inhibitory effect even in the presence of AII (Fraser et al. 2001). Most of the tyrosine phosphoproteins showing changes in capacitated spermatozoa were the same as those identified in uncapacitated cells, but those of ~50 and 70 kDa could be seen to differ in their degree of phosphorylation only in capacitated suspensions, similar to the findings of Adeoya-Osiguwa & Fraser (2000). This suggests that these proteins may have specific functions relating to capacitation and preparation for the acrosome reaction. At present the identity of individual phosphoproteins is unclear but other studies have identified proteins of similar size. In particular, a phosphoprotein of ~95 kDa has been identified in several investigations, including those of Leyton & Saling (1989), Duncan & Fraser (1993), Kalab et al.(1994) and Adeoya-Osiguwa & Fraser (2000). Although Leyton & Saling proposed that this protein is the sperm receptor for ZP3, Kalab et al. reported that it is a hexokinase isoform, its size being 116 kDa in reducing conditions and 95 kDa in non-reducing conditions. Proteins of both sizes were detected in the present study, consistent with results obtained by Adeoya-Osiguwa & Fraser (2000). In addition to these, a phosphoprotein of ~82 kDa has been identified as a kinase anchoring protein 82 (APKAP82) (Carrera et al. 1994) and it is localized mainly in the fibrous sheath of the flagellum.

The present results confirm the hypothesis that AII, like FPP and calcitonin, elicits responses in mammalian spermatozoa by stimulating AC/cAMP, with the increased cAMP then contributing to stimulation of tyrosine phosphorylation. However, unlike FPP and calcitonin, AII stimulated cAMP and phosphorylation in both uncapacitated and capacitated cells. Although the responses to FPP and calcitonin appear to involve G protein-mediated regulation of AC activity, the precise mechanism by which AII is able to stimulate cAMP production remains unclear. The AT1 receptor for AII belongs to the G protein-coupled receptor family (Vinson et al. 1997). In somatic cells, AT1 receptors are usually linked to G{alpha}q-containing G proteins; consequently, AII stimulation is often linked with phospholipase C activation, resulting in increased generation of diacylglycerol and inositol trisphosphate, activation of protein kinase C (PKC) and elevated intracellular Ca2+ concentration (Sayeski et al. 1998). Again in somatic cells, AII has also been reported to activate di-hydropyridine-sensitive Ca2+ channels and to inhibit AC activity (e.g. Ohnishi et al. 1992). However, the responses observed in spermatozoa do not appear to correlate with those obtained in somatic cells. AII clearly stimulates, rather than inhibits, AC/cAMP in mouse spermatozoa. In other studies on human spermatozoa, activation of phosphoinositide-specific phospholipase C and PKC could be detected only in capacitated cells, downstream of opening of dihydropyridine-sensitive Ca2+ channels, as steps in the sequence leading to the acrosome reaction (O’Toole et al. 1996a,b,c). Also, although immunolocalization studies provided evidence for the presence of G{alpha}q/11 in both mouse and human spermatozoa, the location differs somewhat from that of the AT1 receptors, especially in the head: patchy staining for G{alpha}q/11 was observed along the flagellum, at the neck and in the head, particularly in the equatorial segment region (Baxendale & Fraser 2003a), while AT1 appears to be primarily in the acrosomal cap region of the head (Fig. 1Go).

Two recent studies have reported an AII-associated rise in intracellular Ca2+ in mouse (Wennemuth et al. 1999) and equine (Sabeur et al. 2000) spermatozoa. Such a rise could account for the increase in cAMP since AC activity in mouse spermatozoa is stimulated by a rise in available Ca2+ (Fraser & Monks 1990) and membrane-associated AC isoforms (mACs) are known to differ in their Ca2+ requirements (Defer et al. 2000). Mammalian spermatozoa appear to have several isoforms of mAC, some of which are located in the same regions as the AT1 receptors (Baxendale & Fraser 2003b); furthermore, there is evidence suggesting that at least two mACs, with differing Ca2+ requirements, function in spermatozoa (Adeoya-Osiguwa & Fraser 2003). Alternatively, an AII-mediated rise in intra-cellular Ca2+ might stimulate the soluble AC (sAC), molecularly unrelated to mACs, that has been identified in mammalian spermatozoa (Buck et al. 1999). Although sAC is apparently not regulated by G proteins, it is stimulated by both HCO– 3 (Buck et al. 1999) and Ca2+ (Jaiswal & Conti 2003). Further investigation is required to determine more precisely the mechanism whereby AII stimulates cAMP production in mammalian spermatozoa.

In conclusion, in vitro experiments have shown that AII significantly stimulates cAMP production in mouse spermatozoa; the increased availability of cAMP results in significant stimulation of protein tyrosine phosphorylation in both the head and the flagellum. These events could promote changes in the head needed to support the acrosome reaction and changes in the flagellum to promote hyperactivated motility, both of which are required for successful fertilization (Fraser et al. 2001). Western blotting revealed that AII stimulation results in greater phosphorylation of several specific tyrosine phosphoproteins, with two being seen only in capacitated suspensions. Since AII is present in seminal plasma and comes in contact with spermatozoa at the time of ejaculation, it is plausible that AII may play a role in stimulating capacitation in vivo but the unregulated stimulation of cAMP by AII could eventually lead to spontaneous acrosome reactions. This is biologically undesirable, but other seminal plasma molecules with specific receptors on spermatozoa (FPP, calcitonin, adenosine) can regulate cAMP production in the presence of AII and so inhibit spontaneous acrosome loss. Thus contact with seminal plasma exposes mammalian spermatozoa to a number of small molecules that can affect the gametes in biologically significant ways; the acceleration of capacitation and subsequent inhibition of the acrosome reaction would provide a mechanism to ensure that most or all of the relatively few male gametes reaching the site of fertilization in vivo would retain their fertilizing potential and so increase the chance of successful fertilization.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This work was supported by a grant to L R F from the Biotechnology and Biological Sciences Research Council. We thank Drs Susan Adeoya-Osiguwa and Rhona Baxendale for stimulating and constructive suggestions regarding this study. We also thank Kate Kirwan and Mark Simon of the Imaging and Photographic Services Unit in the Randall Centre for Molecular Mechanisms of Cell Function, King’s College London, for their kind help with preparation of figures.


   Footnotes
 
Received 28 October 2003
First decision 23 January 2004
Accepted 25 February 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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