Molecular Human Reproduction, Vol. 7, No. 3, 245-253,
March 2001
© 2001 European Society of Human Reproduction and Embryology
Testis and spermatogenesis |
Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function*
1 Endocrinology and Reproduction Research Group, School of Biomedical Sciences, King's College London, Guy's Campus, London Bridge, London SE1 1UL, 2 Histopathology, St George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, and 3 Biomedical Sciences, Queen Mary Westfield College, Mile End Road, London E1 4NS, UK
Abstract
Fertilization-promoting peptide (FPP) regulates the adenylyl cyclase (AC)/cAMP pathway to elicit capacitation-dependent responses, stimulating capacitation in uncapacitated spermatozoa and then arresting it in capacitated cells, thereby inhibiting spontaneous acrosome reactions. Like FPP, calcitonin and angiotensin II are found in seminal plasma and so might affect sperm function; this study investigated responses in uncapacitated and capacitated mouse spermatozoa to these three peptides. Both calcitonin (5 ng/ml) and angiotensin II (1 and 10nmol/l), like FPP (100nmol/l), significantly stimulated capacitation, assessed using chlortetracycline (CTC) fluorescence and fertilization in vitro analyses. Combinations of two or three peptides, at high and low, non-stimulatory concentrations, were more stimulatory than the individual peptides, suggesting that they may act on the same signalling pathway, plausibly AC/cAMP; preliminary data indicate that calcitonin does stimulate cAMP production. In capacitated cells, FPP and calcitonin elicited pertussis toxin-sensitive inhibition of spontaneous acrosome loss, suggesting involvement of inhibitory G proteins; angiotensin II had no detectable effect. When all three peptides were used, angiotensin II did not interfere with inhibitory responses to FPP/calcitonin. These results suggest that angiotensin II, calcitonin and FPP may somehow modulate the AC/cAMP signal transduction pathway, but the precise mechanisms involved have yet to be elucidated.
acrosome reaction/cAMP/adenylyl cyclase/capacitation/fertilization-promoting peptide
Introduction
Although mammalian spermatozoa are known to be non-fertilizing at the time of release from the male reproductive tract (Austin, 1951
; Chang, 1951), permissive conditions will allow the spermatozoa to `switch on' physiologically whether in the female reproductive tract or under laboratory conditions in vitro. These functionally competent cells are considered to be `capacitated' (Austin, 1952) because they have the capacity to fertilize oocytes, being able both to express hyperactivated motility and to undergo an oocyte-induced acrosome reaction. This acquisition of fertilizing potential, or capacitation, involves many changes to the sperm surface, as well as intracellular components (for review, see de Lamirande et al., 1997), but there is still little information about regulation of these events.
Recent in-vitro studies have revealed that fertilization-promoting peptide (FPP; pGlu-Glu-ProNH2), produced by the prostate gland and found in seminal plasma of several mammals (Cockle, 1995), is a plausible modulator of capacitation in vivo. FPP has been shown to elicit capacitation-dependent responses in vitro in mammalian spermatozoa (Green et al., 1994, 1996a,b; Funahashi et al., 2000), initially stimulating capacitation and fertilizing ability in uncapacitated cells and then arresting capacitation in capacitated cells, resulting in inhibition of spontaneous acrosome reactions. Evidence suggests that once capacitation is initiated, it will continue unchecked, often culminating in the spontaneous acrosome reaction. Biologically, this is undesirable since acrosome-reacted spermatozoa are not able to fertilize intact oocytes (Yanagimachi, 1994). Therefore, these responses to FPP could provide a biologically important mechanism for ensuring that most, or all, of the spermatozoa reaching the site of fertilization in vivo are capacitated but remain acrosome-intact, thus retaining fertilizing potential.
Calcitonin is a 32 amino acid peptide hormone whose chief function is the regulation of Ca2+ fluxes and metabolism. Of the three main phylogenetic classes of calcitonin, i.e. teleost/avian, artiodactyl and rat/human (Pozvek et al., 1997), the first is the most potent, with salmon calcitonin being widely used both in vivo and in vitro (to treat human metabolic bone disorders; see Pozvek et al., 1997). Calcitonin is produced mainly by parafollicular cells in the thyroid; although several somatic tissues have been shown to possess calcitonin receptors, the primary target tissue for calcitonin has been assumed to be bone since calcitonin inhibits osteoclast activity. However, high concentrations of calcitonin in seminal plasma (apparently of prostatic origin; Davis et al., 1989) have been reported, with the mean concentration (~2 ng/ml) being ~40 times that found in human serum (Sjöberg et al., 1980). Early studies carried out in the 1980s reported that: (i) salmon calcitonin inhibited human sperm motility in vitro (Gnessi et al., 1984); (ii) calcitonin bound particularly to the flagellum (Foresta et al., 1986); and (iii) [125I]-salmon calcitonin bound to human spermatozoa, suggesting the presence of calcitonin receptors (Silvestroni et al., 1987). Whether calcitonin might play an important role in modulating sperm function was unknown.
In separate and unrelated investigations, evidence has suggested that the renin–angiotensin system (RAS) may play a role in modulating sperm function. In particular, angiotensin II type-1 (AT1) receptors have been detected on developing spermatids and on mature rat spermatozoa and both AT1 and AT2 subtypes are now known to be present on human spermatozoa (Vinson et al., 1995; Köhn et al., 1998). Angiotensin II, an 8 amino acid peptide whose chief function is the regulation of cardiovascular and electrolyte homeostasis (Vinson et al., 1997), was reported to enhance specific parameters of sperm motility, including those associated with hyperactivation (Vinson et al., 1996). These results may be subject to some ambiguity, however, since measurements were made on cells at room temperature and in the presence of slightly diluted seminal plasma. Recent results (O'Mahony et al., 2000) indicate that human seminal plasma contains immunoreactive angiotensin II at concentrations 5–10-fold higher than those in circulating plasma taken at the same time, the large majority of this being angiotensin II itself. Therefore, endogenous angiotensin II would have been present during the above motility evaluations. Further evidence that the RAS may play a role in promoting male fertility has been obtained from evaluating mice with non-functional genes for the somatic and the testis-specific angiotensin-converting enzyme (ACE) forms. In the latter, males show reduced fertility, despite having normal testes, spermatozoa and mating behaviour (Krege et al., 1995). Although sperm motility was apparently normal (Esther et al., 1996), further investigation indicated that ACE gene disruption resulted in poor sperm transport within the oviduct and poor binding to the zona pellucida (Hagaman et al., 1998). More recently, reduced fertility was reported in both male and female mice genetically deficient for angiotensinogen, the precursor molecule for angiotensin II (Tempfer et al., 2000). Thus evidence from a number of different sources have suggested that angiotensin II plays an, as yet, ill-defined role in normal fertility.
This study was designed to use a well-characterized in-vitro capacitation/fertilization system (Fraser, 1993) to investigate: (i) responses in uncapacitated epididymal mouse spermatozoa to calcitonin and angiotensin II, used individually, with FPP serving as the positive control; (ii) responses in capacitated cells to calcitonin and angiotensin II, again with FPP as the positive control; and (iii) responses obtained using combinations of peptides.
Materials and methods
Media and reagents
The medium used in all experiments was a modified Tyrode's medium (Fraser, 1993) containing 4 mg/ml bovine serum albumin (BSA). All reagents were obtained from Sigma (Poole, Dorset, UK), unless otherwise specified. 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, divided into aliquots and frozen; these stock solutions were kept for a maximum of 1 month. Concentrated stock solutions of angiotensin II and calcitonin were prepared in medium, divided into aliquots, frozen and kept at –20° C until needed. Pertussis toxin stock solution was prepared in distilled water and kept in the cold; for use, the solution was mixed well and diluted with culture medium daily as needed. In all experiments, working stock solutions were 50x the final concentration desired.
Sperm suspension preparation
The contents of cauda epididymides from mature TO male mice (Harlan Olac, Bicester, UK) were released into medium in 30 mm sterile culture dishes (Nunc, Roskilde, Denmark) and allowed to disperse for 5 min. For experimental protocols using uncapacitated spermatozoa, cells were then filtered through short columns of Sephadex G-25 (medium grade; Pharmacia, Uppsala, Sweden) to remove non-motile cells and any epididymal debris. Aliquots of filtered suspensions were then transferred to small (0.5 ml) plastic microcentrifuge tubes, treated as described in Results, gassed briefly with 5% CO2, 5% O2, 90% N2, capped and incubated at 37°C for 40 min. In protocols using capacitated cells, suspensions were incubated for 90 min, then filtered and treated as above.
In all experiments, a small drop of each suspension was examined briefly for motility evaluation. None of the treatments had a deleterious effect on motility and, indeed, treatments of uncapacitated suspensions resulted in more vigorous motility than observed in untreated controls.
Chlortetracycline fluorescence analysis
Chlortetracycline (CTC) analysis was carried out as described previously (Green et al., 1994). An Olympus BX40 microscope equipped with phase contrast and BX-FLA epifluorescence optics using the wide blue–violet excitation cube (U-MWBV) was used for assessments. The excitation beam was passed through a 400–440 nm band pass filter and CTC fluorescence was observed through a DM 455 dichroic mirror. At least 100 spermatozoa in each sample (50 on each of two slides) were classified as expressing on of the three following patterns: F = fluorescence over the entire head, a pattern that is characteristic of uncapacitated, acrosome-intact cells; B = a fluorescence-free band in the postacrosomal region, a pattern that is characteristic of capacitated, acrosome-intact cells; AR = dull or absent fluorescence over the entire sperm head, a pattern that is characteristic of acrosome-reacted cells.
IVF
Ovulation was induced by i.p. injections of 7.5 IU equine chorionic gonadotrophin (Folligon; Intervet, Cambridge, UK) into mature female TO mice followed by 5 IU human chorionic gonadotrophin (HCG, Chorulon; Intervet), ~50 h later. Oviducts were removed ~15 h after HCG administration, and cumulus masses were released into medium covered with autoclaved liquid paraffin (Boots, Nottingham, UK). Sperm suspensions were prepared by releasing epididymal spermatozoa into control medium (contents of two caudal epididymides into 1 ml medium); after 5 min dispersal time, aliquots were removed and stock solutions of peptides were added to give the desired final concentration (see Results for details regarding each experimental series). After 40 min preincubation, sperm suspensions were diluted ~10 fold in medium of the same composition used for the preincubation to give a final concentration of ~2x106 cells/ml; 400 µl were transferred to culture dishes and covered with liquid paraffin. After 65 min co-incubation with sperm suspensions, oocytes were transferred to small droplets of control medium and then fixed at 75 min with buffered formalin (4% formaldehyde in phosphate-buffered saline). Oocytes were stained with 0.75% aceto–orcein, a coverslip was added and oocytes were assessed. A fertilized oocyte (i.e. a zygote) was one that had resumed the second meiotic division and contained a decondensing sperm head.
Statistical analysis
Data were analysed using Cochran's modification of the 
2 test (Snedecor and Cochran, 1980). Because this test involves comparison within individual replicate experiments, consistent patterns of response are required in order to obtain significant differences.
Results
Series I: effect of calcitonin on uncapacitated mouse spermatozoa
Sperm suspensions were prepared as detailed above, divided into aliquots in small microcentrifuge tubes and treated with (final concentration): no additions (control), 100 nmol/l FPP (positive control), 5 ng/ml salmon calcitonin (Sa-CT) or 200 ng/ml human calcitonin (Hu-CT) for 40 min. Three replicate experiments were carried out (n = 3). Through visual examination, sperm motility appeared to be more vigorous in suspensions treated with calcitonin or FPP than in untreated controls. Cells were then stained with CTC, fixed and evaluated. All three treatments significantly stimulated capacitation (P < 0.01; Figure 1) as demonstrated by many fewer F pattern uncapacitated cells and many more capacitated B pattern cells, while there was no detectable effect on the spontaneous acrosome reaction. In the last two replicates, an additional sample was treated with the lower concentration of 20 ng/ml human calcitonin; this treatment also stimulated capacitation but to a slightly lesser extent than the higher concentration of the human hormone (data not shown).
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), the B pattern (
) and the AR pattern (These results suggested that calcitonin treatment would also stimulate fertilizing ability in vitro. This hypothesis was tested by preincubating sperm suspensions for 40 min with no additions (control), 5 ng/ml salmon calcitonin or 200 ng/ml human calcitonin, then diluting them 10 fold in medium of the same composition and adding unfertilized oocytes (n = 4). After fixation, staining and assessment of oocytes, results (Table I
) revealed that both calcitonin treatments significantly stimulated fertilizing ability (salmon calcitonin, P < 0.025; human calcitonin, P < 0.05) compared with untreated controls. The variability in the proportion of fertilized oocytes among the replicates here reflects the use of suspensions that have not fully capacitated and so individual sperm suspensions exhibit different capacitation kinetics; in some, capacitation is quite slow (e.g. only 7% of oocytes fertilized) but in others, it is more rapid (e.g. 60% of oocytes fertilized). The crucial fact is that calcitonin-treated suspensions were more fertile than untreated suspensions.
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Series II: effect of angiotensin II on uncapacitated mouse spermatozoa
Sperm suspensions were prepared, filtered, divided into subsamples and then incubated in the presence of 1 nmol/l and 10 nmol/l angiotensin II, 100 nmol/l FPP (positive control) or nothing (untreated control) for 40 min (n = 3). In suspensions treated with angiotensin II or FPP, sperm motility appeared to be more vigorous than in untreated controls. Capacitation was significantly stimulated (P < 0.01) by both concentrations of angiotensin II and by FPP, but there was no detectable effect on the acrosome reaction (Figure 2
).
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These results suggested that angiotensin II-treated suspensions would be more fertile in vitro than untreated controls. After preincubation of suspensions treated with nothing (control), 100 nmol/l FPP or 10 nmol/l angiotensin II for 40 min, suspensions were diluted 10-fold in medium of the same composition as used for initial incubation and unfertilized oocytes were added (n = 3). Both FPP and angiotensin II significantly stimulated fertilization to the same extent (P < 0.025), compared with controls (Table II
), with both treatments leading to 55–57% of oocytes being fertilized compared with only ~20% in the untreated control group.
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Series III: effect of simultaneous use of calcitonin and angiotensin II on uncapacitated suspensions
Since both calcitonin and angiotensin II significantly stimulated capacitation, responses to combinations of both high concentrations (those used in series I and II) and low concentrations (one-tenth of the high concentrations) were investigated. Sperm suspensions were prepared and incubated for 40 min in the presence of: no additions (control), 5 ng/ml salmon calcitonin (high), 1 nmol/l angiotensin II (high), 5 ng/ml salmon calcitonin + 1 nmol/l angiotensin II (high), 0.5 ng/ml salmon calcitonin (low), 0.1 nmol/l angiotensin II (low) or 0.5 ng/ml calcitonin + 0.1 nmol/l angiotensin II (low; n = 3).
Consistent with series I and II, CTC analysis revealed that high concentrations of calcitonin and angiotensin II significantly stimulated capacitation (P < 0.025) when used individually (Figure 3), but when combined, the stimulation was even higher (P < 0.01). Neither of the low concentrations of calcitonin and angiotensin II had a significant effect when used individually yet, when combined, they significantly stimulated (P < 0.025) capacitation but did not stimulate the spontaneous acrosome reaction.
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Series IV: effect on uncapacitated suspensions of FPP in combination with calcitonin or angiotensin II
The responses to calcitonin and angiotensin II, individually and in combination, were very similar to those observed earlier with FPP, adenosine and FPP + adenosine (Green et al., 1994
, 1996b). Therefore, responses to low FPP in combination with low concentrations of calcitonin and/or angiotensin II were investigated. Sperm suspensions were prepared and treated with: no additions (control), 100 nmol/l FPP (high; positive control), 12.5 nmol/l FPP (low, non-stimulatory; Green et al., 1994), 12.5 nmol/l FPP + 0.5 ng/ml salmon calcitonin (low), 12.5 nmol/l FPP + 0.1 nmol/l angiotensin II (low) or 12.5 nmol/l FPP + 0.5 ng/ml calcitonin + 0.1 nmol/l angiotensin II (low; n = 3). The positive and negative FPP controls elicited the responses previously reported (Green et al., 1994), with 100 nmol/l FPP significantly stimulating capacitation (P < 0.01) while low FPP (12.5 nmol/l) did not, when compared with untreated controls (Figure 4). The combination of low FPP plus either low calcitonin or low angiotensin II significantly stimulated (P < 0.025) capacitation, compared with controls, and the combination of all three low concentrations of peptides was even more stimulatory (P < 0.01).
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In a separate set of experiments, the effect of combining high concentrations of all three peptides was investigated. Sperm suspensions were treated with: no additions (control), 100 nmol/l FPP or 100 nmol/l FPP + 5 ng/ml salmon calcitonin + 1 nmol/l angiotensin II (n = 3). High FPP significantly stimulated capacitation (P < 0.01), but the combination of all three was even more stimulatory (P < 0.001), compared with untreated controls (Figure 5
), with ~75% of cells expressing the B pattern of CTC fluorescence (capacitated, acrosome-intact).
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Series V: effect of calcitonin and angiotensin II on capacitated spermatozoa
Given that calcitonin and angiotensin II significantly stimulated capacitation in uncapacitated mouse spermatozoa, it seemed important to determine whether these peptide hormones had any effect on capacitated cells. As detailed above, FPP, which has similar stimulatory effects to calcitonin and angiotensin II on uncapacitated spermatozoa, also acts on capacitated cells, arresting capacitation by inhibiting spontaneous acrosome reactions (Green et al., 1996b
).
A preliminary experiment revealed that both FPP and calcitonin inhibited spontaneous acrosome reactions in capacitated cells, while angiotensin II did not. Earlier investigations had demonstrated that responses in capacitated spermatozoa to FPP could be blocked by the inclusion of pertussis toxin, suggesting the involvement of inhibitory G proteins (Fraser and Adeoya-Osiguwa, 1999). Therefore, in the subsequent replicates (n = 3), untreated sperm suspensions were preincubated for 90 min, filtered and then incubated for 40 min following addition of: no treatment (control), 100 nmol/l FPP, 100 nmol/l FPP + 100 ng/ml pertussis toxin, 5 ng/ml salmon calcitonin, 5 ng/ml salmon calcitonin + 100 ng/ml pertussis toxin, 1 nmol/l angiotensin II or, finally, 100 nmol/l FPP + 5 ng/ml salmon calcitonin + 1 nmol/l angiotensin II.
100 nmol/l FPP significantly inhibited spontaneous acrosome reactions (P < 0.01), as did 5 ng/ml calcitonin, but the inclusion of pertussis toxin abolished responses to both peptides (Figure 6). In contrast, angiotensin II had no inhibitory effect on spontaneous acrosome reactions. When all three peptides were used simultaneously, the incidence of spontaneous acrosome loss was as low as that seen in suspensions treated with only FPP or calcitonin, indicating that angiotensin II was not able to interfere with responses to FPP and calcitonin.
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Discussion
FPP elicits capacitation-dependent responses in mouse (Green et al., 1994, 1996b) and boar spermatozoa (Funahashi et al., 2000). These responses to FPP involve modulation of the adenylyl cyclase (AC)/cAMP signal transduction pathway (Adeoya-Osiguwa et al., 1998; Fraser and Adeoya-Osiguwa, 1999). Changes in the phosphorylation state of tyrosine residues present in a number of proteins have been detected (Adeoya-Osiguwa and Fraser, 2000), with cAMP production and protein tyrosine phosphorylation being stimulated in uncapacitated cells, but inhibited in capacitated cells.
Given the presence of both calcitonin and angiotensin II in seminal plasma and specific receptors for those two peptides on spermatozoa, the present study investigated responses in vitro of epididymal mouse spermatozoa to the peptides, using FPP as a positive control for comparison. Results indicated that there are, in fact, unexpected similarities in responses to these three peptides.
When assessed using CTC fluorescence, both salmon and human calcitonin significantly stimulated capacitation, suggesting that calcitonin would also stimulate fertilizing ability; subsequent IVF experiments confirmed this. Visual evaluation of suspensions indicated that sperm motility per se in the presence of calcitonin was even better than in untreated controls, and the greater fertilizing ability of spermatozoa treated with both salmon and human calcitonin indicated that hyperactivated motility was stimulated. Successful fertilization requires spermatozoa to express hyperactivated motility; computer-assisted analysis of FPP-treated cells has confirmed hyperactivation (Green et al., 1996c), consistent with their enhanced fertilizing ability (Green et al., 1994). In contrast, other authors (Gnessi et al., 1984) reported that salmon calcitonin, but not human calcitonin, at a concentration of 
4 nmol/l, inhibited human sperm motility in vitro. The concentrations of calcitonin used in this study (~1.5 nmol/l salmon calcitonin; ~60 nmol/l human calcitonin) were not markedly different from those used by Gnessi et al., suggesting that their observations may have been artefactual. Consistent with the demonstrably higher potency of teleost calcitonin, compared with rat and human calcitonin (Pozvek et al., 1997), higher concentrations of human calcitonin were required to obtain results similar to those obtained with salmon calcitonin.
Calcitonin receptors are typical seven transmembrane domain, G protein-coupled, surface-located receptors (Peroutka, 1994). Binding of calcitonin to its receptor has been observed to elicit a range of responses in somatic cells, but the most common response is a stimulation of AC activity with a consequent rise in cAMP (e.g. Siga et al., 1994). Such a mechanism of action would be plausible for the effects of calcitonin on spermatozoa since AC/cAMP is known to play a pivotal role in capacitation (e.g. Fraser and Monks, 1990; de Lamirande et al., 1997).
Angiotensin II also significantly stimulated capacitation in uncapacitated sperm suspensions evaluated with CTC. The magnitude of response obtained was similar with both 1 and 10 nmol/l peptide, indicating that once optimal stimulation has been achieved, higher concentrations will not elicit further response. In an earlier study, equivalent stimulation was obtained with both 100 and 500 nmol/l FPP (Green et al., 1994). The CTC results suggested that angiotensin II-treated suspensions would be more fertile in vitro and this was confirmed by IVF experiments. As with calcitonin, angiotensin II-treated suspensions were highly motile and the enhanced fertilizing ability indicating a stimulation of hyperactivated motility. Earlier observations of human spermatozoa, made in the presence of diluted seminal plasma now known to contain endogenous angiotensin II, had also detected stimulatory effects of exogenous angiotensin II on motility (Vinson et al., 1995, 1996). In the present study, epididymal mouse spermatozoa, having had no prior contact with peptide of seminal plasma origin, were more fertile following a short preincubation in exogenous angiotensin II than untreated controls. These results augment those of Vinson et al. (1995) providing unequivocal evidence that angiotensin II can affect sperm function, including motility, in biologically relevant ways.
Identification of angiotensin II's mechanism of action in spermatozoa is equivocal at present. Like calcitonin receptors, AT1 and AT2 angiotensin receptors are members of the seven transmembrane receptor family (Vinson et al., 1997). AT1 receptors in somatic cells are usually linked to Gq-containing G proteins; consequently angiotensin II stimulation is often associated with phospholipase C activation, leading to increased diacylglycerol and inositol trisphosphate generation, activation of protein kinase C (PKC) and elevation of intracellular Ca2+ [Ca2+]i (Sayeski et al., 1998.). There is also evidence that angiotensin II can activate dihydropyridine-sensitive Ca2+ channels and inhibit AC activity (e.g. Ohnishi et al., 1992). However, activation of phosphoinositide-specific phospholipase C and PKC has only been detected in capacitated human spermatozoa, both being stimulated by an influx of Ca2+ due to opening of dihydropyridine-sensitive Ca2+ channels that also appear to function only in capacitated cells (O'Toole et al., 1996a,b,c). Finally, it seems unlikely that angiotensin II inhibits AC/cAMP since the peptide has been shown to augment responses to FPP. Given that FPP is known to stimulate cAMP production in uncapacitated cells (Adeoya-Osiguwa et al., 1998; Fraser and Adeoya-Osiguwa, 1999), inhibition of cAMP production by angiotensin II would cancel out the stimulatory effect of FPP, not augment it.
Two recent studies, (Wennemuth et al., 1999; Sabeur et al. 2000) which looked at mouse and equine spermatozoa respectively, have reported a rise in intracellular Ca2+ ([Ca2+]i) in cells treated with angiotensin II. A rise in [Ca2+]i could have a general stimulatory effect on a number of important systems in uncapacitated spermatozoa, including AC/cAMP (Fraser and Monks, 1990). However, the precise mechanism of action of angiotensin II in spermatozoa is unclear at present and requires further investigation.
The fact that combinations of calcitonin and angiotensin II, especially at low, non-stimulatory concentrations, had a significant effect on capacitation suggests that both may be working on the same signalling pathway. If they were activating different pathways, then it seems unlikely that low concentrations, in combination, would produce a significant stimulatory effect since individually neither elicited detectable responses. Similarly, the significant effect elicited by a low, nonstimulatory concentration of FPP plus either low calcitonin or low angiotensin II argues for activation of the same pathway. We therefore propose that calcitonin and angiotensin II, like FPP, somehow modulate the AC/cAMP pathway and we now have preliminary (unpublished) data demonstrating that calcitonin does significantly stimulate cAMP production in uncapacitated cells.
Although all three peptides had similar effects on uncapacitated cells, differences were observed with capacitated spermatozoa: both FPP and calcitonin inhibited spontaneous acrosome reactions but angiotensin II did not. Furthermore, responses to FPP and calcitonin were abolished by the inclusion of pertussis toxin, suggesting the involvement of inhibitory G proteins. FPP-treated capacitated mouse spermatozoa have significantly less cAMP than controls, but the inclusion of pertussis toxin along with FPP results in cAMP concentrations similar to those observed in untreated controls (Fraser and Adeoya-Osiguwa, 1999). Indeed, those data suggest that unregulated cAMP production may lead to, or even, promote spontaneous acrosome loss. The present results are consistent with the hypothesis that calcitonin also modulates AC/cAMP in a capacitation-dependent, G-protein mediated manner. In contrast, angiotensin II appears to act only on uncapacitated spermatozoa. There was no evidence for a response in capacitated cells to angiotensin II, used individually or in combination with FPP and calcitonin. The absence of increased acrosome reactions when all three peptides were used indicated that angiotensin II was not able to interfere with the inhibitory effects of FPP and calcitonin on the spontaneous acrosome reaction.
In recent years, a pertussis toxin-sensitive inhibitory G protein has been implicated in the zona pellucida-stimulated acrosome reaction (e.g. Endo et al., 1987; Lee et al., 1992). On the other hand, both the present study and an earlier one (Fraser and Adeoya-Osiguwa, 1999) have indicated that pertussis toxin-sensitive inhibitory G proteins are involved in inhibiting spontaneous acrosome reactions. These hypotheses are not incompatible, since inhibitory G proteins can interact with many different receptors. The continued presence of FPP and calcitonin did not interfere with fertilizing ability, indicating that inhibition of spontaneous acrosome loss did not interfere with a spermatozoon's intrinsic fertilizing ability. In a recent study (Fraser and Adeoya-Osiguwa, 1999), several different inhibitory G
subunits were identified in mouse spermatozoa, raising the possibility that one or some of these subunits may be involved in inhibiting spontaneous acrosome reactions, while others may be involved in the zona-stimulated acrosome reaction.
The present results, indicating that angiotensin II acts only on uncapacitated mouse spermatozoa, differ considerably from those obtained in three recent studies, all reporting that angiotensin II stimulates the acrosome reaction (human, Köhn et al., 1998; bovine, Gur et al., 1998; equine, Sabeur et al., 2000). However, the experimental protocols used were not sufficiently rigorous to support this conclusion unequivocally. In the first study (Köhn et al., 1998), angiotensin II reportedly induced acrosome reactions in human spermatozoa incubated for 3–6 h in the continuous presence of 1–100 nmol/l angiotensin II; pertussis toxin had no effect on the acrosome reaction. Continuous presence of the peptide makes it difficult to conclude that the acrosome reaction was `induced'. In the second study (Gur et al., 1998), a Ca2+-deficient medium was used to `capacitate' bovine spermatozoa, with Ca2+ only being added during the last 20 min; angiotensin II reportedly had no effect on uncapacitated cells but induced the acrosome reaction in capacitated cells (determined by acrosin activity in the supernatant). Because Ca2+ is required for complete capacitation (e.g. de Lamirande et al., 1997), the precise physiological state of the cells is unclear; this makes interpretation of results difficult. In the third study, (Sabeur et al., 2000) the suspensions were preincubated for 3.5 h in the presence of 500 µmol/l 8 Br-cAMP, then angiotensin II was added at 1–1000 nmol/l and a higher proportion of acrosome-reacted spermatozoa was observed in the treated groups than in the untreated controls. The continuous presence of exogenous cAMP makes interpretation difficult.
Each of the above protocols could well result in unregulated increases in cAMP concentrations and recent studies have suggested that this can lead to over-capacitation and triggering of spontaneous acrosome reactions (Fraser and Adeoya-Osiguwa, 1999). In the study of Köhn et al. (1998), the extended incubation under conditions favoring a rise in [Ca2+]i could lead to a rise in cAMP. In the study of Gur et al. (1998), angiotensin II-mediated increases in [Ca2+]i could stimulate cAMP production. Finally, in the study of Sabeur et al. (2000), spermatozoa incubated in medium containing exogenous cAMP would almost certainly have elevated intracellular cAMP levels and addition of angiotensin II could stimulate production of endogenous cAMP, thereby crossing a cAMP threshold that results in spontaneous acrosome loss.
In conclusion, the present study has revealed that FPP, calcitonin and angiotensin II, three unrelated peptides found in seminal plasma, can modulate mammalian sperm function in vitro in biologically important ways. Calcitonin, like FPP, can regulate sperm function in a capacitation-dependent manner, first stimulating capacitation and demonstrable fertilizing ability in vitro, but then arresting capacitation and thereby inhibiting spontaneous acrosome loss. In contrast, angiotensin II stimulated capacitation but did not subsequently arrest it. Combinations of any two or all three peptides, whether used at low or high concentrations, were more effective than peptides used singly. Given that spermatozoa would come into contact with these peptides at ejaculation, it is plausible that they play an important role in regulating mammalian sperm function in vivo, as well as in vitro. Although little is known about mechanisms controlling sperm function in vivo, very few sperm reach the site of fertilization. These peptides could play a crucially important role in the female tract, first stimulating capacitation and then holding the spermatozoa in a state of readiness until an oocyte arrives.
Based on data obtained in the present study, we propose that FPP, calcitonin and [Ca2+]i angiotensin II all act in some way on the same signal transduction pathway, arguably AC/cAMP. By stimulating this pathway in uncapacitated spermatozoa, these peptides would stimulate capacitation, as demonstrated by both CTC analysis and significantly increased fertilizing ability. If these testable hypotheses prove to be correct, then there is considerable redundancy in the mechanisms available to modulate sperm the AC/cAMP signal transduction pathway, further emphasizing the importance of this pathway in mammalian sperm physiology.
Acknowledgments
This study was funded in part by an allocation from the KinetiQue Biomedical Seed Fund of King's College London and Queen Mary Westfield College.
Notes
* This study has previously been published in part, see Fraser et al. (1999). 
4 To whom correspondence should be addressed. E-mail: lynn.fraser{at}kcl.ac.uk
References
Adeoya-Osiguwa, S.A. and Fraser, L.R. (2000) Fertilization promoting peptide and adenosine, acting as first messengers, regulate cAMP production and consequent protein tyrosine phosphorylation in a capacitation-dependent manner. Mol. Reprod. Dev., 63, 384–392.
Adeoya-Osiguwa, S.A., Dudley, R., Hosseini, R. and Fraser, L.R. (1998) FPP modulates mammalian sperm function via TCP-11 and the adenylyl cyclase/cAMP pathway. Mol. Reprod. Dev., 51, 468–476.[Web of Science][Medline]
Austin, C.R. (1951) Observations on the penetration of the sperm into the mammalian egg. Aust. J. Sci. Res. B, 4, 581–596.
Austin, C.R. (1952) The `capacitation' of the mammalian sperm. Nature, 170, 326.[Medline]
Chang, M.C. (1951) Fertilizing capacity of spermatozoa deposited in the fallopian tubes. Nature, 168, 697–698.[Medline]
Cockle, S.M. (1995) Fertilization promoting peptide: a novel peptide, structurally similar to TRH, with patent physiological activity. J. Endocrinol., 146, 3–8.
Davis, N.S., DiSant'Agnese, P.A., Eving, J.F. and Mooney, R.A. (1989) The neuroendocrine prostate: characterization and quantitation of calcitonin in the human gland. J. Urol., 142, 884–888.[Web of Science][Medline]
de Lamirande, E., Leclerc, P. and Gagnon, C. (1997) Capacitation as a regulatory event primes spermatozoa for acrosome reaction and fertilization. Mol. Hum. Reprod., 3, 175–194.
Endo, Y., Lee, M.A. and Kopf, G.S. (1987) Evidence for the role of a guanine nucleotide-binding regulatory protein in the zona pellucida-induced mouse sperm acrosome reaction. Dev. Biol., 119, 210–216[Web of Science][Medline]
Esther, C.R., Howard, T.E., Marino, E.M. et al. (1996) Mice lacking angiotensin-converting enzyme have low blood pressure, renal pathology, and reduced male fertility. Lab. Invest., 74, 953–965.[Web of Science][Medline]
Foresta, C., Caretto, A., Indino, M. et al. (1986) Calcitonin in human seminal plasma and its localization on human spermatozoa. Andrologia, 18, 470–473.[Web of Science][Medline]
Fraser, L.R. (1993) In vitro capacitation and fertilization. Methods Enzymol., 239–253.
Fraser, L.R. and Adeoya-Osiguwa, S.A. (1999) Modulation of adenylyl cyclase by FPP and adenosine involves stimulatory and inhibitory adenosine receptors and G proteins. Mol. Reprod. Dev., 53, 459–471.[Web of Science][Medline]
Fraser, L.R. and Monks, N.J. (1990) Cyclic nucleotides and mammalian sperm capacitation. J. Reprod. Fertil., 42 (Suppl.), 9–21.
Fraser, L.R., Pondel, M.D. and Vinson, G.P. (1999) Calcitonin, angiotensin II and FPP significantly modulate mouse sperm function. J. Reprod. Fertil. (Abstr. Ser.), 24, 8.
Funahashi, H., Asano, A., Fujiwara, T. et al. (2000) Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in boar spermatozoa in vitro. Mol. Reprod. Dev., 55, 117–124.[Web of Science][Medline]
Gnessi, L., Silvestroni, L., Baffri, A. et al. (1984) Salmon calcitonin inhibits human sperm motility in vitro. Biochem. Biophys Res. Commun., 125, 199–204.[Web of Science][Medline]
Green, C.M, Cockle, S.M., Watson, P.F. and Fraser, L.R. (1994) Stimulating effect of pyroglutamylglutamylprolineamide, a prostatic, TRH-related tripeptide, on mouse sperm capacitation and fertilizing ability in vitro. Mol. Reprod. Dev., 38, 215–221.[Web of Science][Medline]
Green, C.M., Cockle, S.M., Watson, P.F. and Fraser, L.R. (1996a) Fertilization promoting peptide, a tripeptide similar to thyrotrophin-releasing hormone, stimulates the capacitation and fertilizing ability of human spermatozoa in vitro. Hum. Reprod., 11, 830–836.
Green, C.M., Cockle, S.M., Watson, P.F. and Fraser, L.R. (1996b) A possible mechanism of action for fertilization promoting peptide, a TRH-related tripeptide that promotes capacitation and fertilizing ability in mammalian spermatozoa. Mol. Reprod. Dev., 45, 244–252.[Web of Science][Medline]
Green, C.M., Cockle, S.M., Watson, P.F. and Fraser, L.R. (1996c) The effect of FPP on the motility characteristics of mouse spermatozoa. J. Reprod. Fertil. (Abstr. Ser.), 17, 31.
Gur, Y., Breitbart, H., Lax, Y. et al. (1998) Angiotensin II induces acrosomal exocytosis in bovine spermatozoa. Am. J. Physiol., 275, E87–93.
Hagaman, J.R., Moyer, J.S., Bachman, E.S. et al. (1998) Angiotensin-converting enzyme and male fertility. Proc. Natl Acad. Sci. USA, 95, 2552–2557.
Köhn, F.-M., Müller, C., Drescher, D. et al. (1998) Effect of angiotensin converting enzyme (ACE) and angiotensins on human sperm functions. Andrologia, 30, 207–215.[Web of Science][Medline]
Krege, J.H., John, S.W, Langenback, L.L. et al. (1995) Male-female differences in fertility and blood pressure in ACE-deficient mice. Nature, 375, 146–148.[Medline]
Lee, M.A., Check, J.H. and Kopf, G.S. (1992) A guanine nucleotide-binding regulatory protein in human sperm mediates acrosomal exocytosis induced by the human zona pellucida. Mol. Reprod. Dev., 31, 78–86.[Web of Science][Medline]
Ohnishi, J., Ishido, M., Shibata, T. et al. (1992) The rat angiotensin II AT1A receptor couples with three different signal transduction pathways. Biochem. Biophys Res. Commun., 186, 1094–1101.[Web of Science][Medline]
O'Mahony, O.H., Djahanbahkch, O., Mahmood, T. et al. (2000) Angiotensin II in human seminal fluid. Hum. Reprod., 15, 1345–1349.
O'Toole, C.M.B., Roldan, E.R.S. and Fraser, L.R. (1996a) Protein kinase C activation during progesterone stimulated acrosomal exocytosis in human spermatozoa. Mol. Hum. Reprod., 2, 921–927.
O'Toole, C.M.B., Roldan, E.R.S. and Fraser, L.R. (1996b) A role for Ca2+ channels in the signal transduction pathway leading to acrosomal exocytosis in human spermatozoa. Mol. Reprod. Dev., 45, 204–211.[Web of Science][Medline]
O'Toole, C.M.B., Roldan, E.R.S. and Fraser, L.R. (1996c) A role for diacylglycerol in human sperm acrosomal exocytosis. Mol. Hum. Reprod., 2, 317–326.
Peroutka, S.J. (1994) Handbook of Receptors and Channels. Vol. 1. G Protein-Coupled Receptors. CRC Press, Boca Raton, FL, USA.
Pozvek, G., Hilton, J.M., Quiza, M. et al. (1997) Structure/function relationships of calcitonin analogues as agonists, antagonists, or inverse agonists in a constitutively activated receptor cell system. Mol. Pharmacol., 51, 658–665.
Sabeur, K., Vo, A.T. and Ball, B.O. (2000) Effects of angiotensin II on the acrosome reaction in equine spermatozoa. J. Reprod. Fertil., 120, 135–142.[Abstract]
Sayeski, P.P., Ali, M.S., Semeniuk, J. et al. (1998) Angiotensin II signal transduction pathways. Reg. Pep., 78, 19–29.
Siga, E., Champigneulle, A. and Imbert-Teboul, M. (1994) cAMP-dependent effects of vasopressin and calcitonin on cytosolic calcium in rat CCD. Am. J. Physiol., 267, F354–365.
Silvestroni, L., Menditto, A., Frajese, G. and Gnessi, G. (1987) Identification of calcitonin receptors in human spermatozoa. J.Clin. Endocrinol. Metabol., 65, 742–746.
Sjöberg, H., Arver, S. and Bucht, E. (1980) High concentration of immunoreactive calcitonin of prostatic origin in human semen. Acta Physiol. Scand., 110, 101–102.[Web of Science][Medline]
Snedecor, G. and Cochran, W. (1980) Statistical methods. Iowa State University Press, Ames, IO, USA.
Tempfer, C.B., Moreno, R.M. and Gregg, A.R. (2000) Genetic control of fertility and embryonic waste in the mouse: a role for angiotensinogen. Biol. Reprod., 62, 457–462.
Vinson, G.P., Saridogan, E., Puddefoot, J.R. and Djahanbakhch, O. (1997) Tissue renin–angiotensin systems and reproduction. Hum. Reprod., 12, 651–662.
Vinson, G.P., Puddefoot, J.R., Ho, M.M. et al. (1995) Type 1 angiotensin II receptors in rat and human sperm. J. Endocrinol., 144, 369–378.
Vinson, G.P., Mehta, J., Evans, S. et al. (1996) Angiotensin II stimulates sperm motility. Reg. Pep., 67, 131–135.
Wennemuth, G., Babcock, D.F. and Hille, B. (1999) Distribution and function of angiotensin II receptors in mouse spermatozoa. Andrologia, 31, 323–325.[Web of Science][Medline]
Yanagimachi, R. (1994) Mammalian fertilization. In Knobil, E. and Neil, J.D. (eds), The Physiology of Reproduction. 2nd edn. Raven Press, New York, USA, pp. 189–317.
Submitted on August 25, 2000; accepted on December 19, 2000.
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