Centre for Reproduction, Endocrinology and Diabetes, School of Biomedical Sciences, Kings College London, Guys Campus, London Bridge, London SE1 1UL, UK
1 To whom correspondence should be addressed. e-mail: lynn.fraser{at}kcl.ac.uk
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Abstract |
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Key words: cAMP/capacitation/decapacitation factor/fucose/spontaneous acrosome reaction
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Introduction |
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Angiotensin II (AII) is another peptide found in seminal plasma at concentrations higher than those found in blood plasma (OMahony et al., 2000) and known to elicit significant responses in mouse sperm (Fraser et al., 2001
). 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., 1986; Fraser and Adeoya-Osiguwa, 1999
; Adeoya-Osiguwa and Fraser, 2002
, 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, 1999
; Adeoya-Osiguwa and Fraser, 2000
; Fraser et al., 2001
). 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., 2001
).
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., 1996). 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., 1997
). A human homologue of the gene Tcp11 has been identified (Ragoussis et al., 1992
) and molecular characterization indicates that the deduced human protein sequence is shorter than, but highly homologous to, the mouse protein (Ma et al., 2002
). 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., 1986
; Silvestroni et al., 1987
) and AII receptors have also been found (Vinson et al., 1995
). 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.
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Materials and methods |
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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 Kings 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 Womens Clinic, the Bridge Centre and the Assisted Conception Unit at Guys 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., 1990). The 95, 70 and 50% v/v Percoll solutions were prepared as described in Green et al. (1996
) 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. (1996) 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 mares 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 12 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 2.53x105 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 Cochrans modification of the 2-test (Snedecor and Cochran, 1980
); 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.
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Results |
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Calcitonin at 0.515 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. (1984) 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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AII had a significant effect at concentrations of 0.1100 nmol/l (Figure 2), with very similar responses being observed with 0.3100 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 0.3100 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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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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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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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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Discussion |
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As mentioned in the Introduction, earlier work had provided some evidence for the presence of calcitonin receptors in human sperm (Foresta et al., 1986; Silvestroni et al., 1987
). In a more recent study using immunolocalization, Adeoya-Osiguwa and Fraser (2003
) 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. (1984
) 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., 2001
). It seems likely that those earlier observations were artefactual, perhaps indicating the presence of some contaminant; the reagents used by Gnessi et al. (1984
) 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., 1997
).
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 FPPs effects on both mouse (Green et al., 1994
) and human sperm (Green et al., 1996
), 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, 2003
). In somatic cells the calcitonin receptor modulation of adenylyl cyclase activity is known to involve G proteins (Pondel, 2000
) 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., 2001
).
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., 2001). 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., 2001
).
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, 1998). Because earlier work had demonstrated that mouse DF can bind to and decapacitate human sperm, as determined by CTC (DasGupta et al., 1994
), 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 DFs 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, 1996
). Unrestrained production of cAMP will stimulate sperm eventually to over-capacitate and undergo the acrosome reaction (Fraser and Adeoya-Osiguwa, 1999
).
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., 2001). In contrast, several papers published fairly recently (bovine, Gur et al., 1998
; human, Köhn et al., 1998
; equine, Sabeur et al., 2000
) 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, 2003). Furthermore, in the present study when we deliberately left a portion of donor semen samples for 23 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., 2000a). 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., 2001
; Lundin et al., 2001
; Fenwick et al., 2002
; Neuber et al., 2003
).
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.
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Acknowledgements |
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References |
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Submitted on September 5, 2003; resubmitted on October 21, 2003; accepted on November 11, 2003.