Human sperm responses to calcitonin, angiotensin II and fertilization-promoting peptide in prepared semen samples from normal donors and infertility patients

Lynn R. Fraser1 and Olufunmilayo O. Osiguwa

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

1 To whom correspondence should be addressed. e-mail: lynn.fraser{at}kcl.ac.uk


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
BACKGROUND: Fertilization-promoting peptide (FPP), angiotensin II (AII) and calcitonin, present in seminal plasma, have significant effects on mouse sperm function in vitro. This study investigated responses of uncapacitated and capacitated human sperm to these peptides, initially using samples from donors with normal semen parameters and then samples from men attending infertility clinics. METHODS: Prepared suspensions were incubated in the presence/absence of a range of peptide concentrations and assessed using chlortetracycline (CTC) analysis and the hamster oocyte penetration test. RESULTS: In uncapacitated suspensions, maximal stimulatory responses (CTC) were obtained with calcitonin at 0.5–15 nmol/l and AII at 0.3–100 nmol/l; FPP is known to be most effective at 100 nmol/l. All peptides also significantly stimulated sperm penetrating ability. Combinations of peptides at low concentrations, having no detectable effect when used singly, elicited significant responses, suggesting that they work via the same signalling pathway. In suspensions incubated in the presence of fucose to accelerate capacitation and acrosome reactions, both FPP and calcitonin, but not AII, inhibited acrosome loss; however, AII did not interfere with responses to FPP and calcitonin. Unlike samples from 15 donors, some samples from >70 patients had high proportions of capacitated and/or acrosome-reacted cells when assessed immediately following preparation. Even so, the peptides usually elicited responses similar to those obtained with donor samples and combinations of peptides inhibited spontaneous acrosome loss for at least 3 h. CONCLUSIONS: The responses obtained in vitro suggest that these peptides could have significant effects on human sperm function in vivo and could also be used effectively in infertility clinics.

Key words: cAMP/capacitation/decapacitation factor/fucose/spontaneous acrosome reaction


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
It has been known for over half a century that mammalian sperm are non-fertilizing at the time of release from the male reproductive tract, being required to undergo post-release maturational changes, termed ‘capacitation’ (Austin, 1952Go), in order to become functional (Austin, 1951Go; Chang, 1951Go). Despite extensive research into capacitation, much is still unknown about the molecular events that underpin it. However, in the past decade, a number of studies have provided evidence that several small molecules present in seminal plasma can bind to specific receptors on mammalian sperm and elicit significant capacitation-dependent responses that regulate sperm function. In human seminal plasma, fertilization-promoting peptide (FPP) is found at ~50 nmol/l (Fraser and Adeoya-Osiguwa, 2001Go), adenosine is found in micromolar concentrations (Fabiani and Ronquist, 1995Go), and calcitonin is found at ~2 ng/ml, ~40-fold that in human serum (Sjöberg et al., 1980Go). All three molecules have been shown to stimulate capacitation and demonstrable fertilizing ability in uncapacitated mouse sperm, while in capacitated cells they inhibit spontaneous acrosome loss (Fraser and Adeoya-Osiguwa, 2001Go); FPP and adenosine have also been shown to act similarly on porcine sperm (Funahashi et al., 2000aGo,b). Although both responses are biologically important, the latter is particularly so since acrosome-reacted sperm, even if highly motile, are unable to fertilize intact unfertilized oocytes (Yanagimachi, 1994Go).

Angiotensin II (AII) is another peptide found in seminal plasma at concentrations higher than those found in blood plasma (O’Mahony et al., 2000Go) and known to elicit significant responses in mouse sperm (Fraser et al., 2001Go). Like FPP and calcitonin, it was found to stimulate capacitation and fertilizing ability in uncapacitated cells, but unlike the other two peptides, AII did not inhibit spontaneous acrosome reactions. However, when combinations of FPP and/or calcitonin plus AII were added to capacitated mouse sperm, spontaneous acrosome loss was inhibited. Thus, the inhibitory responses elicited by FPP and calcitonin are not reversed by AII in mouse sperm.

FPP, adenosine and calcitonin have been shown to regulate the adenylyl cyclase (AC)/cAMP signalling pathway, initially stimulating and then inhibiting cAMP production (Stein et al., 1986Go; Fraser and Adeoya-Osiguwa, 1999Go; Adeoya-Osiguwa and Fraser, 2002Go, 2003). Furthermore, these responses are G protein-mediated, as demonstrated by the ability of pertussis toxin to inhibit responses to these molecules in capacitated suspensions (Fraser and Adeoya-Osiguwa, 1999Go; Adeoya-Osiguwa and Fraser, 2000Go; Fraser et al., 2001Go). Since combinations of low concentrations of AII plus either FPP or calcitonin were shown to have a significant stimulatory effect on uncapacitated mouse sperm, it was hypothesized that AII also acts on AC/cAMP but in an indirect manner, perhaps by causing a rise in intracellular Ca2+; this would be consistent with AII only having a stimulatory effect (Fraser et al., 2001Go).

The first aim of the present study was to use human semen samples from donors with normal semen parameters to test the hypothesis that responses to FPP, calcitonin and AII in both uncapacitated and capacitated suspensions would be similar to those obtained using mouse sperm. This was plausible, given the evidence for the presence of the requisite specific receptors on human sperm. FPP had already been shown to stimulate capacitation in uncapacitated human cells, indicating the presence of FPP receptors (Green et al., 1996Go). TCP11, the putative receptor for FPP, is located on the acrosomal cap region of acrosome-intact mouse sperm and on the flagellum, especially the principal piece (Fraser et al., 1997Go). A human homologue of the gene Tcp11 has been identified (Ragoussis et al., 1992Go) and molecular characterization indicates that the deduced human protein sequence is shorter than, but highly homologous to, the mouse protein (Ma et al., 2002Go). TCP11 distribution on human sperm is similar to that seen on mouse gametes (M.Tahmasebi, personal communication). Earlier studies provided indirect evidence, based on calcitonin binding to human sperm, that calcitonin receptors are present (Foresta et al., 1986Go; Silvestroni et al., 1987Go) and AII receptors have also been found (Vinson et al., 1995Go). Since those initial results supported the hypothesis being tested, the second aim of the study was to assess responses of sperm from patients attending infertility clinics to determine whether the peptides were effective on these samples and whether the effect could be maintained for several hours.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Media and reagents
Unless otherwise stated, all reagents were obtained from Sigma (Poole, UK). The standard medium used was Earle’s medium with added penicillin (100 IU/ml) and human serum albumin (HSA) at 4 mg/ml. For the hamster oocyte penetration test (HOPT), a modified Biggers–Whitten–Whittingham (BWW) medium (Biggers et al., 1971Go) with 20 mmol/l HEPES and bovine serum albumin at 3 mg/ml was used. FPP solutions were prepared as described earlier (Green et al., 1994Go), lyophilized and stored at –20°C. Samples were reconstituted in HSA-free medium, divided into aliquots and frozen; these were used within 1 month. Concentrated stock solutions of salmon calcitonin and human angiotensin II were prepared in medium, divided into aliquots, frozen and maintained at –20°C until needed. In all experiments, working stock solutions were x50 the desired final concentration. For the HOPT, progesterone was added to sperm suspensions to trigger the acrosome reaction in capacitated cells. A stock solution of 10 mg/ml progesterone was prepared in dimethylsulphoxide (DMSO) and diluted to 1 mg/ml using 1:1 DMSO:BWW; this was used at 1 in 100 to give a final concentration of 10 µg/ml.

Sperm suspension preparation
For the initial investigations, healthy donors, all found to have ‘normal’ semen parameters (defined for our experiments as having a concentration ≥40x106/ml, ≥50% motile and 50% with reasonable morphology determined subjectively on fresh samples), provided semen samples obtained by masturbation. In total we had 15 donors, including five known-fertile men (four of whom were retired donor insemination donors). The use of human semen samples for this project received ethical approval from the King’s College Research Ethics Committee. In the later investigations, the remainder of patients’ semen samples, provided for evaluation just prior to initial consultation at the London Women’s Clinic, the Bridge Centre and the Assisted Conception Unit at Guy’s Hospital, were evaluated. The Ethics Committees of each of the three units approved the use of these samples for this project and all patients gave written consent for this use of their leftover semen samples. A research licence (licence no. R0195/-1) was obtained from the Human Fertilisation and Embryology Authority (HFEA) to carry out the HOPT.

Motile sperm were obtained using mini-Percoll gradients (Ord et al., 1990Go). The 95, 70 and 50% v/v Percoll solutions were prepared as described in Green et al. (1996Go) and discontinuous gradients were made using 300 µl of each. After centrifugation for 5 min at 600 g, pelleted cells were removed, resuspended in fresh medium, centrifuged again for 5 min at 600 g and resuspended in fresh medium. The sperm concentration was determined using a haemocytometer and adjusted to 5x106 cells/ml. A small drop of each prepared suspension was placed on a slide and motility was examined; usually >90% of cells exhibited progressive motility. In the rare instances where motility in a prepared sample was poor, the sample was not used. Suspensions were transferred to 10 ml centrifuge tubes, relevant peptides were added and cells were incubated at 37°C in an atmosphere of 5% CO2, 5% O2, 90% N2.

Cell assessment
The live/dead status of each spermatozoon was assessed using the vital dye Hoechst bis-benzimide 33258 and the functional status was assessed using the chlortetracycline (CTC) fluorescence assay. The methodology described by Green et al. (1996Go) for preparation of reagents, treatment of suspensions, preparation of slides and assessment of cells was used. An Olympus BX41 microscope [Olympus Optical Co. (UK) Ltd, UK] equipped with phase contrast and epifluorescent optics was used to evaluate the cells, first with the U-MWU2 fluorescence cube (wide ultraviolet) to assess live/dead status and then the U-MWBV2 fluorescence cube (wide blue-violet) to assess CTC patterns. In each sample, 100 live cells were assessed for CTC patterns. The three main patterns of CTC fluorescence observed were: F, with uniform fluorescence over the head, characteristic of uncapacitated, acrosome-intact cells; B, with a fluorescence-free band in the postacrosomal region, characteristic of capacitated, acrosome-intact cells; AR, with dull or absent fluorescence over the sperm head, characteristic of capacitated, acrosome-reacted cells. In most samples, there were very few dead cells (0 to <5%); in the rare instances when there were many, the data were not used.

Hamster oocyte penetration test
Mature female hamsters (Charles River UK Ltd, UK) were injected with 25 IU pregnant mare’s serum gonadotrophin (Folligon; Intervet, Cambridge, UK) and, 52 h later, with 25 IU hCG (Chorulon; Intervet); 18 h later the females were killed, the oviducts were removed and the cumulus masses were released into BWW medium. Cumulus cells were removed by treating the oocytes with medium containing 1 mg/ml hyaluronidase (Type III from sheep testes) for ~1–2 min. Cumulus-free oocytes were washed once in BWW medium and then incubated in BWW containing 1 mg/ml trypsin (Type I from bovine pancreas) for ~2 min, until the zona could be seen to be dissolving. Oocytes were removed immediately and washed twice in fresh BWW medium; when sperm suspensions were ready, oocytes were transferred into them.

Sperm suspensions were prepared in BWW, treated as described in Results for Series III and diluted to give a final concentration of 2.5–3x105 cells/ml. After gamete co-incubation, oocytes were transferred into fresh medium to remove loosely adhering sperm, transferred to another drop of fresh medium and fixed by adding buffered formalin. Fixed oocytes were stained with 0.75% aceto-orcein, mounted and assessed for penetration; the number of decondensing sperm heads in each oocyte was determined.

Statistical analysis
Both CTC and HOPT results were analysed using Cochran’s modification of the {chi}2-test (Snedecor and Cochran, 1980Go); with this test, there must be consistent responses of sufficient magnitude in individual replicates in order for the responses to be significant. Each treated sample was compared with the appropriate control sample.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Series I: Does calcitonin accelerate capacitation in uncapacitated human sperm?
To determine whether calcitonin had an effect on uncapacitated human sperm, sperm suspensions were prepared as described above, divided into aliquots, and calcitonin at 0.015, 0.15, 0.5, 1.5 and 15 nmol/l was added to separate aliquots; suspensions receiving no peptide served as the untreated control and those receiving FPP at 100 nmol/l served as the positive control. Suspensions were incubated for 1 h, stained with Hoechst 33258 and CTC, then fixed; five replicate suspensions were evaluated (n = 5).

Calcitonin at 0.5–15 nmol/l significantly (P < 0.025 to P < 0.01) stimulated capacitation (Figure 1); all three concentrations evaluated in this range appeared to be equally effective, while the two lowest concentrations had no detectable effect. The response to calcitonin was similar in magnitude to that obtained with FPP, the positive control. Although Gnessi et al. (1984Go) had reported that salmon calcitonin depressed human sperm motility, we observed good motility in all incubated samples, with and without salmon calcitonin.



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Figure 1. Human sperm responses to salmon calcitonin. At concentrations ≥0.5 nmol/l, calcitonin significantly stimulated capacitation in uncapacitated human sperm suspensions treated for 1 h and then analysed using chlortetracycline (CTC). Treatments were: none (Con), 100 nmol/l fertilization-promoting peptide (FPP) (positive control) and 0.015–15 nmol/l salmon calcitonin (Cal). Data are presented as % cells (mean ± SEM; n = 5) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. **P < 0.025, ***P < 0.01 compared with untreated control suspensions.

 
Series II: Does angiotensin II accelerate capacitation in human sperm?
To test responses to AII, uncapacitated human sperm suspensions were prepared and then separate aliquots were treated with AII at 0.01, 0.03, 0.1, 0.3, 1, 10 and 100 nmol/l. As before, suspensions receiving no peptide served as the untreated control and those receiving 100 nmol/l FPP served as the positive control. Suspensions were incubated for 1 h, then stained, fixed and assessed (n = 7 for controls and AII at 0.1–1 nmol/l; n = 4 for other concentrations).

AII had a significant effect at concentrations of 0.1–100 nmol/l (Figure 2), with very similar responses being observed with 0.3–100 nmol/l (P < 0.01); 0.1 nmol/l appeared to be on the cusp of biological activity with a lesser, but still significant, response (P < 0.05) than that obtained with the higher concentrations. The degree of stimulation obtained with 0.3–100 nmol/l AII was very similar to that obtained with FPP. As with calcitonin, good motility was maintained in all samples during incubation.



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Figure 2. Human sperm responses to angiotensin II. At concentrations ≥0.1 nmol/l, angiotensin II significantly stimulated capacitation in uncapacitated human sperm suspensions treated for 1 h and then analysed using chlortetracycline (CTC). Treatments were: none (Con), 100 nmol/l fertilization-promoting peptide (FPP) (positive control) and 0.01–100 nmol/l human angiotensin II (AII). Data are presented as % cells (mean ± SEM; n = 5) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. *P < 0.05, ***P < 0.01 compared with untreated control suspensions.

 
Series III: Are peptide-treated human sperm suspensions more effective in the HOPT?
Since CTC analysis of calcitonin- and AII-treated suspensions indicated the presence of more capacitated cells than in the untreated controls, we hypothesized that treated suspensions would be more effective in penetrating zona-free hamster oocytes. Because FPP and calcitonin inhibit spontaneous acrosome loss, the protocol involved preincubating suspensions with or without peptide for 1 h, then adding progesterone for 30 min to induce the acrosome reaction in capacitated cells. Before undertaking the HOPT experiments, we confirmed that sperm responded as predicted (n = 5). As shown in Figure 3, the peptides stimulated capacitation and progesterone stimulated significantly more cells to undergo the acrosome reaction in the peptide-treated suspensions. In an earlier study of human sperm responses to FPP, the same suspensions were used for both the HOPT and CTC evaluataion (Green et al., 1996Go); this dual analysis makes the experiments complicated and we felt it need not be repeated in the present study.



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Figure 3. Progesterone-induced acrosome reactions in human sperm preincubated in the presence of peptides. Uncapacitated human sperm suspensions were preincubated for 1 h with or without peptides, followed by progesterone (P) at 10 µg/ml for 30 min, and then analysed using chlortetracycline (CTC). Initial treatments were: no peptide (Con + P), 100 nmol/l fertilization-promoting peptide (FPP + P), 1.5 nmol/l calcitonin (Cal + P) and 1 nmol/l angiotensin II (AII + P); results from control suspensions incubated for the same total time but without added progesterone (Con) are also shown. Data are presented as % cells (mean ± SEM; n = 5) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. *P < 0.05, **P < 0.025, ***P < 0.01, ****P < 0.001 compared with control suspensions.

 
Sperm suspensions (from the donors known to be fertile; n = 5) were prepared as above in BWW medium and separate aliquots were treated with 100 nmol/l FPP, 1.5 nmol/l calcitonin or 1 nmol/l AII; controls received no peptide. After incubation for 1 h, 10 µg/ml progesterone was added to all suspensions and incubation was continued for 30 min. Suspensions were diluted in BWW to a final concentration of 2.5–3x105 cells/ml, approximately equal numbers of oocytes were added to each suspension and gametes were co-incubated for 3 h. Oocytes were fixed, stained with 0.75% aceto-orcein and examined for the presence of one or more decondensing sperm heads; approximately equal numbers of oocytes were analysed in each treatment group (149–161). Significantly more oocytes (P < 0.025) were penetrated by sperm in the peptide + progesterone-treated suspensions (61–66%) than in the peptide-free + progesterone-treated control suspensions (31%; Figure 4). As further evidence of the functional superiority of peptide-treated suspensions, the incidence of polyspermy obtained with those suspensions was ~44% compared with 29% in the control suspensions; this is consistent with the presence of more acrosome-reacted sperm in peptide-treated suspensions.



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Figure 4. Enhanced penetrating ability of human sperm preincubated in the presence of peptides plus progesterone. Fertilization-promoting peptide (FPP), calcitonin and angiotensin II significantly stimulated penetrating ability in uncapacitated human sperm suspensions, treated for 1 h with/without peptide and then for 30 min with 10 µg/ml progesterone (prog), when tested in the hamster oocyte penetration test. Treatments were: no peptide (Con + prog), 100 nmol/l FPP (FPP + prog), 1.5 nmol/l calcitonin (Cal + prog) and 1 nmol/l angiotensin II (AII + prog). Data are presented as % penetrated oocytes (mean ± SEM; n = 5); the number of penetrated oocytes out of total oocytes in each treatment group is shown above each bar. **P < 0.025 compared with no peptide control suspensions.

 
Series IV: What is the response of uncapacitated human sperm to combinations of peptides?
Combinations of both high (significantly stimulatory) and low (no significant response) concentrations of FPP, calcitonin and AII were evaluated: high FPP = 100 nmol/l, low FPP = 10 nmol/l; high calcitonin = 1.5 nmol/l, low calcitonin = 0.15 nmol/l; high AII = 1 nmol/l, low AII = 0.03 nmol/l. Suspensions were incubated for 1 h, then stained, fixed and assessed (n = 9 for untreated control, 100 nmol/l FPP and all combinations; n = 4 for high and low calcitonin and AII assessed individually).

To simplify presentation of data, the results with high calcitonin and high AII are not shown since the pattern and magnitude of response were similar to that seen in Figures 1 and 2 and to that obtained with FPP in this series (Figure 5). Combinations of high concentrations of calcitonin + AII and FPP + calcitonin + AII produced a significant (P < 0.01) stimulation of capacitation, very similar to that obtained with high FPP. Although the low concentrations of calcitonin and AII elicited no significant response when used individually, in combination they produced a significant (P < 0.01) response; this was also true when low FPP was included.



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Figure 5. Responses to combined peptides at high and low concentrations. Combinations of peptides at both high and low concentrations significantly stimulated capacitation in uncapacitated human sperm suspensions treated for 1 h and then analysed using chlortetracycline (CTC). Treatments were: none (Con), 100 nmol/l fertilization-promoting peptide (FPP) (Hi F; positive control), 1.5 nmol/l calcitonin + 1 nmol/l angiotensin II (Hi C + A), 1.5 nmol/l calcitonin + 1 nmol/l angiotensin II + 100 nmol/l FPP (Hi C + A + F), 0.15 nmol/l calcitonin (Lo C), 0.03 nmol/l angiotensin II (Lo A), 0.15 nmol/l calcitonin + 0.03 nmol/l angiotensin II (Lo C + A) and 0.15 nmol/l calcitonin + 0.03 nmol/l angiotensin II + 10 nmol/l FPP (Lo C + A + F). Data are presented as % cells (mean ± SEM; n = 4–9) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. ***P < 0.01 compared with untreated control suspensions.

 
Series V: What is the effect of these peptides, alone or in combination, on capacitated human sperm?
Although these peptides had been evaluated with capacitated mouse sperm, none had been tried on capacitated human sperm. On the assumption that at least FPP and calcitonin might inhibit spontaneous acrosome loss, it was necessary to develop test conditions that promoted both capacitation and a relatively high incidence of acrosome loss in the control suspensions in order to be able to demonstrate inhibition by peptides. It was desirable to carry out the whole experiment on the same day, but even when suspensions were incubated for several hours, there was not a sufficiently high incidence of spontaneous acrosome reactions. We therefore investigated whether adding fucose to the medium would accelerate capacitation and acrosome loss. Uncapacitated mouse sperm have a decapacitation factor (DF) that binds, via fucose residues, to fucose binding sites on its receptor; the latter is attached to the plasma membrane via a GPI anchor. Endogenous DF can be displaced readily from uncapacitated mouse sperm by exogenous fucose, resulting in capacitated, fertile suspensions (Fraser, 1998Go). The fact that mouse sperm DF can also decapacitate human sperm (DasGupta et al., 1994Go) suggests that human sperm have a similar DF, and we reasoned that exogenous fucose might displace the corresponding human DF and so accelerate capacitation; that proved to be the case. Although the mouse DF can be removed readily from mouse sperm by gentle centrifugation (significant shift from the F to the B pattern of CTC), the human DF appears to be more tightly bound to human sperm since centrifugation during Percoll preparation does not usually result in a majority of cells in the B pattern of CTC.

The new protocol involved adding 10 mmol/l fucose to prepared uncapacitated human sperm suspensions (an untreated control was also included) and incubating them for 2 h; then the suspension was divided and 100 nmol/l FPP, 1.5 nmol/l calcitonin and 1 nmol/l AII were added to separate aliquots. After a further 2 h incubation, cells were stained, fixed and assessed (n = 7). Both untreated controls and fucose-only treated suspensions were evaluated at various time-points. As can be seen in Figure 6, a 4 h incubation in the presence of exogenous fucose significantly accelerated capacitation so that there were fewer uncapacitated and more capacitated cells than in the untreated controls at 4 h and ~37% of sperm had acrosome-reacted, compared with ~21% in the untreated controls. Against this background of elevated acrosome loss, both FPP and calcitonin were seen to significantly (P < 0.05) inhibit the acrosome reaction; the proportion of AR pattern cells at the end of the incubation was essentially the same as at the time peptides were added. In contrast to the other two peptides, AII had no inhibitory effect and ~40% of sperm were acrosome-reacted.



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Figure 6. Inhibition of acrosome reactions in capacitated suspensions by fertilization-promoting peptide (FPP) and calcitonin, but not angiotensin II. Suspensions were incubated in the absence/presence of 10 mmol/l fucose to accelerate capacitation for a total of 4 h, with peptides being present for the last 2 h; cells were then analysed using chlortetracycline (CTC). Treatments were: none (Con), fucose only (Fuc), and fucose + 100 nmol/l FPP (Fuc + F), 1.5 nmol/l calcitonin (Fuc + C) or 1 nmol/l angiotensin II (Fuc + A). Data are presented as % cells (mean ± SEM; n = 7) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. *P < 0.05, **P < 0.025, ***P < 0.01 compared with 4 h fucose-treated suspensions.

 
To determine whether the inhibitory effects of FPP and calcitonin could be abolished by AII, 2 h fucose-treated suspensions had combinations of FPP + AII, calcitonin + AII, or FPP + calcitonin + AII added and incubation was continued for an additional 2 h (n = 5). Assessment of these treatments indicated that inhibition of acrosome loss was maintained (Figure 7), demonstrating that the presence of AII did not interfere with responses to either FPP or calcitonin.



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Figure 7. Inhibition of acrosome reactions by fertilization-promoting peptide (FPP) and calcitonin persists even in the presence of angiotensin II. Suspensions were incubated in the presence of 10 mmol/l fucose for a total of 4 h, with peptides being present for the last 2 h, and cells were then analysed using chlortetracycline (CTC). Treatments were: fucose only (Fuc), fucose + 100 nmol/l FPP + 1 nmol/l angiotensin II (Fuc + F + A), fucose + 1.5 nmol/l calcitonin + 1 nmol/l angiotensin II (Fuc + C + A) and fucose + 100 nmol/l FPP + 1.5 nmol/l calcitonin + 1 nmol/l angiotensin II (Fuc + F + C + A). Data are presented as % cells (mean ± SEM; n = 5) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. *P < 0.05 compared with 4 h fucose-treated suspensions.

 
Series VI: Do sperm in prepared semen samples from patients attending infertility clinics respond to FPP, calcitonin and AII in the same way as sperm from donors?
Having established that FPP, calcitonin and AII significantly affect human sperm function in samples from donors (several of whom were of proven fertility) with ‘normal’ semen parameters, we wanted to evaluate responses in semen samples from men attending infertility clinics at the initial consultation. We arranged to obtain the remainder of samples provided by men for semen analysis on the day of initial consultation at various infertility units; the possible causes of fertility problems in these couples had not been identified. As many of the samples as possible were evaluated, the main constraints being sample volume and sperm concentration. Because we only received what was left of samples after evaluation by the clinic, not all ‘leftovers’ had sufficient volume; we needed to obtain ≥1 ml of semen in order to have enough sperm to assess following Percoll preparation. In addition, only samples with >20x106 cells/ml and reasonable motility would yield enough cells to assess. Almost all of the ~75 samples assessed had a concentration >30x106 cells/ml, >50% motility and reasonable morphology. All samples were evaluated at least at time ‘0’, i.e. as soon as the sample had been prepared. If there were sufficient sperm, then aliquots were treated with no peptide, 100 nmol/l FPP, 1.5 nmol/l calcitonin, 1 nmol/l AII and a combination of all three peptides; after incubation for 1 h, cells were stained, fixed and assessed. In a few samples, there were also sufficient cells to evaluate effects of the combination after 3 h.

It was not practical to show 70 time 0 profiles, so we chose to show a group of samples obtained from the clinic closest to our laboratory; with these, the time from our receipt of samples to their Percoll preparation would have been minimized. Figure 8 shows the starting profile for 29 semen samples from 27 patients, compared with a typical time ‘0’ CTC pattern distribution for our normal donors (Con) where the majority of cells exhibit the F pattern (uncapacitated, acrosome-intact), ~25% show the B pattern (capacitated, acrosome-intact) and relatively few show the AR pattern (capacitated, acrosome-reacted). That distribution was observed consistently during the first part of the study using donor samples (see Figures 1, 2, 5, 6). It was surprising to see that many of the patients’ CTC pattern profiles differed from the distribution observed with sperm from donors; only about six of the 29 samples shown had a distribution similar to that of the donors. Many of the others had a minority of F pattern cells, but a large proportion of either B or AR pattern cells, i.e. these distributions suggest that the sperm were in more advanced stages of capacitation. For two patients, we were able to obtain both the initial and then a second sample, several weeks apart; samples denoted 1* and 2* in Figure 8 are from the same patient and, similarly, 3** and 4** are two independent samples from a different patient. It is interesting to note that in the repeat samples of both patients there was only a minority of F pattern cells, suggesting that the profile is relatively consistent for individual men.



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Figure 8. Chlortetracycline (CTC) profiles for semen samples (n = 29) from 27 different patients following Percoll preparation are variable and most differ markedly from the typical profile seen in samples from normal at time ‘zero’. The values for 1* and 2* represent data obtained with two different samples, several weeks apart, from the same patient; the same applies to 3** and 4**, for a different patient. Data are presented as % cells expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. Statistical evaluation was not possible since each profile is n = 1.

 
Since we had no control over the time elapsing between production of the sample by the patient and our receiving the remainder, we considered the possibility that at least some of these differences might reflect a greater time elapsing between these two events for the patient samples than that between production of samples by donors and our receiving them. While a longer interval could, in theory, tend to skew the distribution towards the capacitated patterns, we would expect this to produce a reasonably consistent change rather than the very wide variation seen. To determine whether the apparent differences between donor and patient CTC profiles were just time-dependent parameters, several donor semen samples were prepared and analysed both (i) immediately and (ii) after 2–3 h maintenance of the neat semen sample in the incubator. As can be seen in Figure 9, after extended holding there was a shift from the uncapacitated F pattern to the capacitated B and AR patterns, although there was no large increase in AR cells; the differences were not statistically significant (n = 5). Therefore, we think that the data from the patients’ samples suggest that some of these patients have sperm that may be physiologically advanced, compared with cells in our donor samples.



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Figure 9. Effect of extended incubation of donor semen samples on chlortetracycline (CTC) patterns. Percoll preparation of sperm was carried out both immediately and after 2–3 h incubation of neat semen samples at 37°C. Data are presented as % cells (mean ± SE; n = 5) expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence. The differences between samples prepared immediately and after holding for 2–3 h were not statistically significant.

 
This aim of this part of the study was to evaluate responses to individual and combined peptides in all samples that had sufficient sperm following Percoll preparation. Statistical analysis of the responses was not possible since each sample was from a different patient; given the wide range of starting points and the lack of diagnoses regarding possible reasons for infertility, there were no grounds for combining different data sets. Therefore, it seemed most appropriate to show the response patterns for selected patients (Figure 10) and to make some general comments. For samples that started out with a majority of B pattern cells, there was very little one could expect to see following peptide treatment. However, we did note that in these samples, as well as in the majority of the others, AII-treated suspensions had more AR pattern cells than in either the Con-1 h sample or those treated with FPP or calcitonin (for example, see Figure 10A, B and D), indicating that AII actively stimulated the acrosome reaction. Despite this, the combined peptide treatment usually suppressed this stimulatory response to AII. Of the individual peptides, FPP was the most consistent to stimulate capacitation and inhibit acrosome loss, and, even if one of the peptides appeared to be ineffective when used individually, the combined peptides usually inhibited the acrosome reaction. Furthermore, in prepared samples with enough cells to extend the treatment, the peptide combination proved to be as effective after 3 h as after 1 h.



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Figure 10. A composite showing responses in samples from four different patients (AD) to individual and combined peptides after 1 h for all and also after 3 h for C and D; these patients are among those whose starting chlortetracycline (CTC) profiles are shown in Figure 7. Treatments were: none (Con), 100 nmol/l fertilization-promoting peptide (F), 1.5 nmol/l calcitonin (C), 1 nmol/l angiotensin II (A) and a combination of all three peptides at these concentrations (Com). Data are presented as % cells expressing the F pattern (open column), B pattern (hatched column) and AR pattern (cross hatched column) of CTC fluorescence.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
FPP, calcitonin and AII are all found in human seminal plasma at concentrations higher than in blood plasma and all have been shown to have biologically important effects on mammalian sperm. Although FPP was already known to stimulate uncapacitated human sperm, nothing was known about responses in capacitated human cells and no in-depth study of responses to either calcitonin or AII had been undertaken. This study was designed to test the hypothesis that all three peptides would elicit responses in human sperm similar to those obtained in mouse sperm. An additional aim was to investigate responses to peptide treatment in sperm obtained from men attending infertility clinics.

As mentioned in the Introduction, earlier work had provided some evidence for the presence of calcitonin receptors in human sperm (Foresta et al., 1986Go; Silvestroni et al., 1987Go). In a more recent study using immunolocalization, Adeoya-Osiguwa and Fraser (2003Go) found calcitonin receptors to be present on both the acrosomal cap and flagellar regions of mouse sperm and a similar distribution has been obtained with human cells (M.Tahmasebi, personal communication). When responses to a range of salmon calcitonin concentrations were evaluated using CTC analysis, concentrations of ≥0.5 nmol/l were found to significantly stimulate capacitation (Figure 1). Essentially the same response was obtained with all the effective concentrations, while the two lowest concentrations had no detectable effect. Although Gnessi et al. (1984Go) reported that salmon calcitonin, but not human calcitonin, inhibited human sperm motility, we could detect no visible negative effect of any of the concentrations on sperm motility. Indeed, calcitonin-treated human sperm would not have been able to penetrate zona-free hamster oocytes if motility had been compromised. Furthermore, salmon calcitonin-treated mouse sperm capacitated more quickly and fertilized mouse oocytes more rapidly than untreated controls, providing evidence that treated cells were able to express hyperactivated motility (Fraser et al., 2001Go). It seems likely that those earlier observations were artefactual, perhaps indicating the presence of some contaminant; the reagents used by Gnessi et al. (1984Go) were the gift of another investigator rather than being purchased from a reliable commercial source such as we have used. Salmon calcitonin was used in the present study because calcitonins from fish are biologically more potent than human calcitonin (Pozvek et al., 1997Go).

Given the presence of AII receptors on human sperm (see Introduction), it was plausible that AII would accelerate capacitation in human cells and the results support this. All concentrations of ≥0.3 nmol/l significantly stimulated capacitation to essentially the same extent as 100 nmol/l FPP, while 0.1 nmol/l was less effective (Figure 2); the two lowest concentrations had no detectable effect. In contrast to these results, each concentration of calcitonin tested either elicited a strong response or none; there was no concentration that produced a modest response, analogous to that obtained with 0.1 nmol/l AII. In earlier studies of FPP’s effects on both mouse (Green et al., 1994Go) and human sperm (Green et al., 1996Go), there was more evidence of a concentration-dependent response, but a fairly narrow range of concentrations was used (each differing from the next by a factor of 2). In the present study we wished to evaluate a wider range of concentrations, in case human cells required more or less than mouse cells, and so most of the concentrations represented a 10-fold step from the adjacent concentrations. Although it would have been possible to identify more precisely the cut-off point for responses, that was not the focus of this study. Evidence that these responses to peptides involve specific receptor comes from recent studies carried out in mouse sperm. Calcitonin has been shown to regulate production of cAMP, initially stimulating cAMP in uncapacitated suspensions and then inhibiting cAMP in capacitated suspensions (Adeoya-Osiguwa and Fraser, 2003Go). In somatic cells the calcitonin receptor modulation of adenylyl cyclase activity is known to involve G proteins (Pondel, 2000Go) and inclusion of pertussis toxin was shown to inhibit the responses of capacitated mouse sperm to both calcitonin and FPP, indicative of G protein involvement (Fraser et al., 2001Go).

Since these CTC results suggested that peptide-treated sperm suspensions would be more fertile than the untreated controls, similarly treated suspensions were tested with zona-free hamster oocytes. Because FPP and calcitonin inhibit the acrosome reaction, and only acrosome-reacted sperm will fuse with the hamster oocyte plasma membrane, suspensions were first incubated with the peptides and then with progesterone to induce the acrosome reaction in capacitated cells (see Figure 3). These suspensions were functionally superior to the no peptide + progesterone control suspensions, with twice as many penetrated oocytes (Figure 4) and a higher incidence of polyspermy. Thus the functional test results supported the conclusions drawn from the CTC data, namely that all three peptides stimulate human sperm fertilizing ability.

The combination of high concentrations of calcitonin and AII produced responses very similar to those obtained with individual peptides (Figure 5), whereas in mouse sperm, such a combination gave greater stimulation (Fraser et al., 2001Go). This probably reflects the differences in the sources of the cells: cauda epididymal mouse sperm that had not contacted seminal plasma versus ejaculated human sperm that were in seminal plasma at the time samples were received. The fact that prepared human sperm suspensions do respond to the different peptides indicates that at least some of the endogenous peptide molecules have been removed during Percoll preparation, but it is plausible that not all were removed, resulting in some receptors being already occupied when exogenous peptide was added. When combinations of low concentrations, ineffective when used singly, were evaluated, a significant response was observed, suggesting involvement of a common signal transduction pathway. As detailed in the Introduction, both FPP and calcitonin have been shown to regulate AC/cAMP in mouse sperm and this is likely to be true for human cells as well. Although the precise mechanism by which AII might stimulate cAMP production has yet to be determined, we have hypothesized that it may cause a rise in intracellular Ca2+ and so stimulate the AC directly (Fraser et al., 2001Go).

In order to determine whether any of the peptides had an effect on spontaneous acrosome reactions in capacitated cells, a protocol that would both accelerate capacitation and push the sperm to undergo the acrosome reaction was developed. Since even extended incubation did not result in a sufficiently high incidence of acrosome reactions to be able to detect significant inhibitory responses, we developed a biologically sound method that worked well. From investigations of a decapacitation factor (DF) present on uncapacitated epididymal mouse sperm, we established that fucose residues on the DF bind to fucose binding sites on the DF receptor (DF-R); furthermore, endogenous mouse DF can be displaced by the addition of exogenous fucose, resulting in a marked acceleration of capacitation, the acrosome reaction and demonstrable fertilizing ability (Fraser, 1998Go). Because earlier work had demonstrated that mouse DF can bind to and decapacitate human sperm, as determined by CTC (DasGupta et al., 1994Go), it was plausible that human cells have a similar DF-R and, therefore, that exogenous fucose would accelerate capacitation. This proved to be the case, as shown in Figure 6. After 4 h in 10 mmol/l fucose, only a small minority of human sperm remained uncapacitated and a large proportion had acrosome-reacted. Thus it was possible to carry out the desired experiments and the results demonstrated that both FPP and calcitonin significantly inhibit acrosome loss, while AII does not. When AII was used in combination with FPP, calcitonin or both, AII did not interfere with the inhibitory responses to the other two peptides (Figure 7). The effectiveness of fucose in stimulating human sperm capacitation is consistent with our current understanding of DF’s mechanism of action. When present, the DF activates a Ca2+ATPase and helps maintain low intracellular Ca2+, but its loss allows intracellular Ca2+ to rise and this can stimulate AC activity and production of cAMP (Adeoya-Osiguwa and Fraser, 1996Go). Unrestrained production of cAMP will stimulate sperm eventually to ‘over-capacitate’ and undergo the acrosome reaction (Fraser and Adeoya-Osiguwa, 1999Go).

Thus, all the in vitro experimental evaluations of human sperm responses to the three peptides indicated that both uncapacitated and capacitated mouse and human sperm respond similarly, with the same concentrations being effective in both. Since the peptides are found in seminal plasma, as well as blood and other body fluids, and human sperm have the appropriate receptors, it is plausible that these peptides could elicit similar regulatory responses in vivo. Of particular importance is the ability of FPP and calcitonin initially to stimulate capacitation and then to inhibit spontaneous acrosome loss, thus preserving fertilizing potential; their ability to maintain the inhibition, even in the presence of AII, is also important. Given that very few sperm reach the uterine tube, successful fertilization would be more likely if the majority of sperm present were capacitated but still acrosome-intact.

Having characterized the responses of human sperm from men with normal semen profiles, we were able to investigate the responses of sperm from patients attending infertility clinics. As noted in the Results, only samples that had acceptable motility and concentration were assessed because methods used for analysis require a reasonable number of prepared motile cells. Thus the samples were not ones that would have been categorized by the clinics as having obvious ‘male factor’ problems. It was therefore very surprising that the CTC patterns observed immediately after Percoll preparation revealed that many of the patients had profiles with a minority of uncapacitated F pattern cells and high proportions of B pattern (capacitated, acrosome-intact) and/or AR pattern (capacitated, acrosome-reacted) cells, compared with profiles routinely observed using samples from our donors. When there was sufficient semen sample to allow evaluation of responses to peptides, a frequently observed feature was stimulation of the acrosome reaction by AII, compared with the untreated control and the FPP- and calcitonin-treated subsamples. This would be consistent with sperm in many of the samples being in an advanced physiological state. In our earlier study on mouse sperm, the addition of AII to capacitated suspensions failed to inhibit acrosome loss, but it did not appear to stimulate it either (Fraser et al., 2001Go). In contrast, several papers published fairly recently (bovine, Gur et al., 1998Go; human, Köhn et al., 1998Go; equine, Sabeur et al., 2000Go) reported that AII stimulates the acrosome reaction. However, the experimental protocols employed in those studies involved either long or complicated incubations in the presence of AII or preincubation in a cAMP analogue followed by AII, making interpretation difficult. The present results suggest that if sperm are quite advanced, then AII can promote a spontaneous acrosome reaction, most likely in an indirect manner by stimulating cAMP production, and this could explain the results obtained in the above published studies.

Looking at responses to FPP and calcitonin, FPP was the more consistently effective and, in the majority of samples, the combination of all three peptides was effective in both stimulating capacitation and inhibiting acrosome loss; this was true even when AII used singly stimulated acrosome loss. Furthermore, in the few samples where it was possible to evaluate responses to the combined peptides at both 1 and 3 h, the inhibition of spontaneous acrosome reactions was maintained (see Figure 10 for examples).

Because these patterns were unexpected, we considered whether they might be artefactual, possibly reflecting the greater length of time between sample production and our receiving it. This time lag was unavoidable since the clinics were not able to release the remainder of individual samples until evaluations had been completed. Although it is possible that the time factor made some contribution to the results obtained, we would predict that the effects would be broadly similar in all the samples, but it is clear from Figure 8 that the starting profiles were quite variable. Furthermore, repeat samples from two patients had profiles very similar to the initial samples. By chance, we obtained a sample produced by a donor used by a clinic for its own sperm bank (so known to be fertile) rather than from a patient. Despite the relatively lengthy journey needed to obtain the sample and then bring it back for analysis, the CTC profile was similar to that of our normal donors, with a majority of cells showing the F pattern characteristic of uncapacitated cells. In contrast, many of the patients’ samples, including those from the clinic providing that donor sample, had a minority of F pattern cells, suggesting accelerated capacitation. It seems unlikely that extended exposure to seminal plasma and the peptides in it would have a marked physiological effect since most of the Ca2+ in seminal plasma is chelated. In a recent study using mouse sperm, the inclusion of EGTA to chelate traces of Ca2+ in Ca2+-deficient medium abolished any responses to calcitonin and FPP (Adeoya-Osiguwa and Fraser, 2003Go). Furthermore, in the present study when we deliberately left a portion of donor semen samples for 2–3 h before preparing it for CTC analysis, the profiles we obtained had clearly shifted somewhat from F to B patterns, but there was no major increase in AR patterns; with n = 5, the differences were not statistically significant (Figure 9). One possible explanation could be that in some samples from patients there was either less DF present initially or DF was less tightly bound and so more susceptible to removal during Percoll preparation.

These peptides are ‘natural’ molecules that human sperm would encounter at ejaculation, and since semen samples used in infertility clinics are almost always prepared so as to remove seminal plasma, we suggest that these peptides could be used effectively with prepared sperm suspensions intended for both intrauterine insemination and IVF. The most consistent responses in the patients’ samples were obtained with the combined peptides: all three stimulate capacitation and then FPP and calcitonin inhibit acrosome reactions, resulting in a build-up of potentially fertilizing sperm. Samples with slow capacitation and/or motility would benefit, as indeed would ‘normal’ samples; with more potentially fertilizing cells in peptide-treated samples, it would be possible to use lower numbers for insemination in vitro and so reduce the incidence of polyspermy. A recent study demonstrated that when pig sperm suspensions were preincubated in the presence of adenosine, which acts in a manner analogous to FPP and calcitonin, it was possible to reduce the number of cells used for insemination and so reduce the incidence of polyspermy, a common problem with IVF in the pig (Funahashi et al., 2000aGo). Sperm that are capacitated and acrosome-intact are ready to fertilize once they contact an oocyte. Rapid penetration would prevent ageing of unfertilized oocytes and thus should result in relatively early embryonic cleavage to the 2-cell stage; there have been several recent reports that early cleaving human embryos have a higher implantation potential when used for embryo transfer following IVF (e.g. Bos-Mikich et al., 2001Go; Lundin et al., 2001Go; Fenwick et al., 2002Go; Neuber et al., 2003Go).

In conclusion, this study has demonstrated that FPP, calcitonin and AII elicit biologically important responses in human sperm in vitro, consistent with earlier evidence that mouse sperm respond in a similar way. Given that all three peptides are present in seminal plasma and human sperm possess receptors for each, it is plausible that the peptides could have similar effects in vivo. This would help to ensure that the relatively few sperm reaching the site of fertilization would be capacitated and acrosome-intact, i.e. potentially fertilizing. The results obtained with semen samples from both donor and infertility patients also suggest that the peptides, especially a combination of all three, could be used effectively in a clinical setting. Their addition to prepared, seminal plasma-free, sperm suspensions would stimulate capacitation but then inhibit spontaneous acrosome reactions, resulting in a high proportion of sperm able to effect rapid fertilization.


    Acknowledgements
 
This study was supported by funds from the Kinetique Biomedical Seed Fund. We are grateful to the staff and patients at the London Women’s Clinic, the Bridge Centre and the Assisted Conception Unit at Guy’s Hospital, as well as our normal donors, for assisting us in this study.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on September 5, 2003; resubmitted on October 21, 2003; accepted on November 11, 2003.