Hum. Reprod. Advance Access originally published online on February 3, 2006
Human Reproduction 2006 21(5):1184-1193; doi:10.1093/humrep/dei486
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Effects of estrogenic xenobiotics on human and mouse spermatozoa
Reproduction and Rhythms Group, School of Biomedical and Health Sciences, King’s College London, London, UK
1 Present address: Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA
2 To whom correspondence should be addressed at: Reproduction and Rhythms Group, School of Biomedical and Health Sciences, King’s College London, London SE1 1UL, UK. E-mail: lynn.fraser{at}kcl.ac.uk
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Abstract |
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OBJECTIVE: To investigate human sperm responsiveness to the estrogenic xenobiotic genistein and seek further information regarding the mechanism of action of estrogenic xenobiotics using mouse spermatozoa. METHODS: Uncapacitated human spermatozoa were incubated with genistein and assessed using chlortetracycline (CTC) fluorescence. CTC was also used to evaluate mouse sperm responses to daidzein and combinations of genistein, 8-prenylnaringenin and nonylphenol. Several steroids were tested to determine structure–function relationships, and possible involvement of cAMP and G proteins in responses was also investigated. RESULTS: Genistein significantly accelerated capacitation and acrosome loss in human spermatozoa, with 1, 10 and 100 nmol/l being equally effective. In mouse spermatozoa, daidzein produced significant responses, and combinations of xenobiotics at low concentrations were more effective than used singly. The compounds appear to act at the cell surface, and responses to three different steroids were nonidentical. A protein kinase-A inhibitor blocked responses to xenobiotics, while genistein and nonylphenol significantly stimulated cAMP production. Pertussis toxin and dideoxyadenosine blocked responses, suggesting involvement of inhibitory G proteins and membrane-associated adenylyl cyclases. CONCLUSION: Human and mouse sperm responses to genistein are very similar, but human gametes appear to be even more sensitive. The mechanism of action may involve unregulated stimulation of cAMP production, leading to significant acrosome loss, undesirable because already acrosome-reacted cells are nonfertilizing. Xenobiotics were even more effective in combination. Since simultaneous exposure to low concentrations of multiple xenobiotics is likely to occur in animals and humans, further investigation is needed to determine whether this could impair fertility.
Key words: membrane-associated adenylyl cyclases/cAMP/G proteins/genistein/infertility
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Introduction |
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During the past several decades, a wide variety of environmental compounds, often termed ‘xenobiotics’, have been implicated in health and reproductive effects in humans and animals. Humans are exposed to a wide range of bioactive flavonoids, including natural phytoestrogens, via the diet (Mazur and Adlercreutz, 2000
; Kris-Etherton et al., 2002) and bioactive synthetic compounds through dietary, household and occupational exposure. The latter include alkylphenol polyethoxylates, phthalates and persistent pollutants such as pesticides. Animals can contact xenobiotics via their food and chemicals used widely in agriculture, such as pesticides, herbicides and fertilizers. The realization that humans and animals are exposed to many potentially bioactive xenobiotics has raised concerns about their possible significance for both health and fertility. Interest has ranged from the possible beneficial effects of phytochemicals on cancer and cardiovascular disease (Kris-Etherton et al., 2002), with some now being promoted as dietary supplements, to adverse effects of industrially derived compounds on development and reproductive function (Sharpe, 2001; Skakkebaek et al., 2001).
Most studies on effects of xenobiotics, especially the ‘endocrine disrupters’, have focused on long-term developmental influences on the testis and male reproductive tract, with reproductive tract abnormalities and semen quality often being assessed as the biologically important end-points. However, we recently provided evidence that very low concentrations of several xenobiotics, all considered to be weakly estrogenic when tested in standard bioassays, have subtle, but biologically extremely relevant, acute and direct effects on the function of mature spermatozoa by significantly accelerating capacitation and the acrosome reaction (Adeoya-Osiguwa et al., 2003). In those studies, the effects of short-term exposure of uncapacitated mouse spermatozoa to genistein (found in soya and other legumes; Mazur and Adlercreutz, 2000), 8-prenylnaringenin (found in hops and beer; Milligan et al., 1999, 2002) and nonylphenol (found in a range of industrial products including paint and pesticides; Sonnenschein and Soto, 1998) were assessed using chlortetracycline (CTC) analysis. Even at low nanomolar concentrations, all three compounds significantly stimulated both capacitation and the acrosome reaction. Genistein and 8-prenylnaringenin were the most potent, with 
10 nmol/l eliciting essentially maximal responses, compared with the 1 µmol/l needed with nonylphenol. Those CTC results suggested that suspensions treated for a short time would be more fertile, and in vitro fertilization experiments confirmed this: mouse spermatozoa pre-incubated for approximately 15 min in 10 nmol/l of either genistein or 8-prenylnaringenin were significantly more fertile in vitro than untreated controls (Adeoya-Osiguwa et al., 2003).
Of particular interest was the finding that these compounds were highly effective at concentrations much lower than the 10 µmol/l required for 17
-estradiol. Furthermore, responses were not blocked by the anti-estrogen hydroxytamoxifen, suggesting that classical estrogen receptors (ERs) were not involved. While genistein is known to have other effects, including protein tyrosine kinase inhibition, the concentrations required for such effects are very much higher (e.g. up to 185 µmol/l: Pukazhenthi et al., 1998; Tomes et al., 2004) than those we found to stimulate capacitation in mouse spermatozoa. Inhibition of tyrosine phosphorylation would result in inhibition of both capacitation and acrosome loss. In contrast, we observed accelerated capacitation, which is associated with enhanced protein tyrosine phosphorylation (Adeoya-Osiguwa and Fraser, 2000) and accelerated acrosome loss when spermatozoa were treated with these xenobiotics. Therefore, it seems highly unlikely that genistein’s stimulatory effects on capacitation are due to inhibition of phosphorylation.
Following on from those initial investigations, the present study was designed to address a number of points. Firstly, we wanted to determine whether human spermatozoa would react to the weakly estrogenic xenobiotics in a manner similar to mouse spermatozoa. This would give some insight into whether such responses were exclusive to mouse gametes or reflected a general sensitivity in mammalian spermatozoa. In addition, if human spermatozoa proved to be responsive in vitro, there might be consequences for fertility in vivo since already acrosome-reacted spermatozoa are nonfertilizing (de Lamirande et al., 1997). We chose to assess genistein because it was very potent in our earlier study, and because it is an environmental compound that many humans and animals may be exposed to as a result of ingesting soya-containing products. In addition, we investigated the effects on mouse spermatozoa of (i) daidzein (another isoflavone found in soya and other legumes), (ii) combinations of low concentrations of xenobiotics and (iii) other steroids (to look for structure–function relationships). Finally, since unregulated cAMP production appears to contribute to increased spontaneous acrosome loss (Fraser et al., 2003), we assessed the possible involvement of cAMP and G proteins in mediating the observed responses. A short abstract of part of this study was published in the abstract book of the 21st Annual Meeting of the European Society of Human Reproduction and Embryology (Fraser et al., 2005).
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Materials and methods |
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Media and reagents
A standard modified Tyrode’s medium (Fraser, 1993
) containing 1.80 mmol/l CaCl2 and 4 mg/ml bovine serum albumin (BSA) was used for mouse sperm suspensions, while Earle’s medium with added penicillin (100 IU/ml) and human serum albumin at 4 mg/ml was used for human sperm suspensions. All reagents were purchased from Sigma (Poole, Dorset, UK) unless otherwise stated. CGS 21680 stock solution was prepared daily at 5 mmol/l in dimethylsulphoxide (DMSO). Fertilization promoting peptide (FPP) was prepared and stored as described by Green et al. (1994). Stock solutions (10 mmol/l) of all steroids, genistein (Gen), 8-prenylnaringenin (8 PN) and nonylphenol (NP) were prepared in absolute ethanol (Analar grade; Merck, Poole, UK), aliquotted and stored at –20°C; stock solutions (5 mmol/l) of daidzein and 2',5'-dideoxyadenosine (ddAdo) (Calbiochem, Merck Biosciences, Beeston, UK) were prepared in DMSO and stored at –20°C. Pertussis toxin stock was made up in sterile distilled water to a concentration of 100 µg/ml and stored in the refrigerator. Working solutions of all test compounds were prepared daily, using standard medium as diluent; all compounds were used at a 1/50 dilution to give desired final concentrations. We have found that the very low final concentrations of nonaqueous solvents present in these experiments have no detectable effect on sperm function.
Sperm suspension preparation
For mouse sperm suspensions, cells were released from the cauda epididymides of mature TO males (Harlan, Bicester, UK) into a sterile dish (Nunc, Roskilde, Denmark) containing modified Tyrode’s medium (approximately 0.8 ml per male); all suspensions contained spermatozoa from at least 2–4 males, depending on the assay. To remove nonmotile cells, suspensions were allowed to disperse for 5 min on a warming tray, then filtered through short columns containing Sephadex G-25 (medium grade; Amersham Biosciences, Little Chalfont, UK), pre-equilibrated with medium. Unlike their human counterparts, mouse spermatozoa should not be centrifuged to obtain motile cells; this removes a decapacitation factor and so promotes accelerated capacitation even before experimental treatment has begun (Fraser, 1984). Filtered suspensions were assessed briefly to check that motility was satisfactory (approximately 90% motile), then treated as detailed in the Results. Suspensions were incubated in an atmosphere of 5% CO2: 5% O2: 90% N2 at 37°C for approximately 35 min, then stained with CTC, fixed and assessed.
Motile human sperm suspensions, obtained from semen samples provided by young normal donors (one known to be fertile, having previously provided samples for successful donor insemination), were prepared using discontinuous Percoll gradient centrifugation, followed by washing and resuspension in Earle’s medium (Fraser and Osiguwa, 2004); the sperm concentration was adjusted to 5 x 106 cells/ml. The use of human semen samples for this research has received ethical approval from the King’s College London Research Ethics Committee. Suspensions were incubated with/without xenobiotic for 1 h in an atmosphere of 5% CO2: 5% O2: 90% N2 at 37°C, then stained with the vital dye Hoechst bis-benzimide 33258 followed by CTC, fixed and assessed (methodology described by Green et al., 1996). Since human spermatozoa generally capacitate more slowly than mouse spermatozoa, incubating suspensions in test compounds for 1 h allows reliable detection of any stimulatory responses (e.g. Fraser and Osiguwa, 2004).
CTC fluorescence analysis
The functional state of both mouse and human spermatozoa was assessed using the CTC fluorescence assay (described in Green et al., 1994, 1996). An Olympus BX41 microscope (Olympus Optical, UK) equipped with phase contrast and epifluorescent optics was used for assessments. Hoechst 33258 analysis of human spermatozoa, to detect dead cells, used the U-MWU2 fluorescence cube (wide UV) and CTC analysis of both human and mouse cells used the U-MWBV2 fluorescence cube (wide blue-violet). In each treated sperm sample in each replicate, 100 cells were classified as having one of three staining patterns: F, with fluorescence over the entire head, characteristic of uncapacitated, acrosome-intact spermatozoa; B, with a fluorescence-free band in the post-acrosomal region, characteristic of capacitated, acrosome-intact spermatozoa; and AR, with dull or absent fluorescence over the entire head, characteristic of acrosome-reacted spermatozoa (see Fraser, 1995 for photographs of CTC patterns in bull, human and mouse spermatozoa). Hoechst 33258 analysis showed that there were very few dead human spermatozoa (<5%). Slides were not scored blind, but more than one individual was involved in obtaining the present CTC results and, from time to time, slides that had been read by one individual were then also read by another. In those instances, evaluations made by the two assessors were similar and consistent.
cAMP assay
The amount of cAMP produced in live, intact mouse spermatozoa was determined using a nonradioactive enzyme immunoassay kit (RPN225) from Amersham Biosciences. Assays, in triplicate or quadruplicate for each treatment in each replicate experiment, were performed as described in the instruction booklet provided with the kit.
Statistical analysis
CTC results were analysed using Cochran’s modification of the chi-square test (Snedecor and Cochran, 1980); this compares responses within each replicate and then sums the values. To obtain a statistically significant difference, the responses must be consistent and the difference in responses between the experimental and control values must be sufficiently large in each replicate. cAMP results were analysed using a paired t-test (Sigma Stats; Jandell Scientific International, Chicago, IL, USA).
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Results |
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Series I: genistein accelerates capacitation and acrosome loss in human spermatozoa
Since our earlier study using mouse spermatozoa investigated responses to 1, 10 and 100 nmol/l genistein (Adeoya-Osiguwa et al., 2003
), these same concentrations were used to test human spermatozoa. Uncapacitated human sperm suspensions were incubated in the absence or presence of 1, 10 and 100 nmol/l genistein for 1 h at 37°C (n = 4). An aliquot receiving 100 nmol/l FPP served as a positive control and significantly accelerated capacitation (P < 0.01), but not acrosome loss (Figure 1A), consistent with earlier studies (Green et al., 1996; Fraser and Osiguwa, 2004). FPP is a tripeptide found in seminal plasma in nanomolar concentrations and has been shown to regulate capacitation, initially stimulating production of cAMP and capacitation, then inhibiting cAMP production and spontaneous acrosome loss (Fraser et al., 2003).
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), B pattern (
) and AR pattern (
) of CTC fluorescence. ***P < 0.01, ****P < 0.001 compared with untreated control suspensions (Con). (B) These are data from the earlier study of Adeoya-Osiguwa et al. (2003)All three concentrations of genistein significantly stimulated (P < 0.01–0.001) both capacitation and acrosome loss to the same extent when compared with the untreated controls (Figure 1A). Interestingly, genistein was more effective than FPP at accelerating capacitation, as shown by the much lower proportion of cells expressing the uncapacitated F pattern of CTC fluorescence in the genistein-treated samples.
For comparison, data from our earlier study (Adeoya-Osiguwa et al., 2003), showing the responses of mouse spermatozoa to genistein, are presented in Figure 1B. As can be seen, at least 10 nmol/l genistein was needed to achieve maximal responses in mouse spermatozoa, but 1 nmol/l was as effective as 10 and 100 nmol/l with human spermatozoa. Furthermore, there were more acrosome-reacted human spermatozoa following treatment with 1–100 nmol/l genistein (30–37% AR pattern cells) than mouse spermatozoa (17–22%). Thus, human and mouse spermatozoa respond to genistein in the same way, but human cells appear to be even more responsive to genistein than their mouse counterparts.
Series II: daidzein, like genistein, accelerates capacitation in mouse spermatozoa
Daidzein and genistein are both isoflavones found in soya and other legumes, so daidzein was tested to determine whether it elicits the same responses as genistein. Uncapacitated mouse spermatozoa were incubated for approximately 35 min in the absence or presence of 10 and 100 nmol/l daidzein; 100 nmol/l genistein was included as the positive control (n = 3). Daidzein proved to be as effective as genistein in both accelerating capacitation and triggering the acrosome reaction (Figure 2).
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Series III: combinations of low concentrations of xenobiotics are more effective than individual compounds on mouse spermatozoa
Uncapacitated mouse spermatozoa were incubated for approximately 35 min in the absence or presence of low concentrations of xenobiotics, used both singly and in combination. In one set of experiments, 1 nmol/l genistein and 1 nmol/l nonylphenol were evaluated, with 10 nmol/l genistein serving as a positive control; 2 nmol/l genistein was included for comparison with the combination treatment (n = 5). In a second set of experiments, genistein and 8-prenylnaringenin were used at both 1 and 0.1 nmol/l, the latter being a concentration not tested previously (n = 4).
As shown in Figure 3, 1 nmol/l genistein used on its own significantly accelerated capacitation, while 1 nmol/l nonylphenol had no significant effect; this is consistent with the differences in potency noted in our earlier study (Adeoya-Osiguwa et al., 2003). When used in combination, however, there was an enhanced effect on capacitation, with significantly fewer uncapacitated F pattern cells and significantly more capacitated B pattern cells than in suspensions treated with only a single compound. The response to this combination was essentially the same as that obtained with 2 nmol/l genistein, suggesting that both compounds are working via the same mechanism of action. Consistent with our earlier study, 10 nmol/l genistein was more effective than either xenobiotic used at 1 nmol/l.
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In the second set, genistein and 8-prenylnaringenin were more effective when used in combination than when used singly (Figure 4). At 1 nmol/l, there were fewer uncapacitated F pattern cells and more B- and AR patterned cells, in the combination treatment suspensions. At the lower concentration of 0.1 nmol/l, each xenobiotic significantly accelerated capacitation and, when used in combination, they also caused a significant increase in acrosome-reacted cells.
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Series IV: several structurally different estradiols accelerate capacitation in mouse spermatozoa
Uncapacitated mouse sperm suspensions were incubated for approximately 35 min in the absence or presence of 10 µmol/l 17-estradiol, 17
-estradiol (less effective in binding to ERs) and BSA-conjugated 17-estradiol, which is unable to cross the plasma membrane (n = 4). Responses to all three compounds were essentially the same, with significant acceleration of capacitation and stimulation of acrosome loss (Figure 5), suggesting that classical intracellular ERs are not involved.
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Series V: mouse sperm responses to different steroids are not identical
Uncapacitated mouse sperm suspensions were incubated for approximately 35 min in the absence or presence of 10 µmol/l 17-estradiol, 5-dihydrotestosterone and progesterone to determine whether the responses observed with estradiol were common to many steroids (n = 6). Interestingly, the responses to each steroid differed. As before, 17-estradiol significantly stimulated both capacitation and acrosome loss. In contrast, 5-dihydrotestosterone significantly accelerated capacitation, but not acrosome loss, while progesterone had no detectable effect (Figure 6). Thus, the response is steroid-specific rather than nonspecific.
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Series VI: xenobiotics significantly stimulate cAMP production in uncapacitated mouse spermatozoa
To test the hypothesis that these responses to xenobiotics might be due to increased cAMP production, uncapacitated mouse sperm suspensions were incubated for approximately 35 min in the absence or presence of 10 µmol/l 17-estradiol or 10 nmol/l genistein ± 20 µmol/l Rp-cAMPS, a metabolically stable, membrane-permeant competitive inhibitor of protein kinase A (n = 3). As expected, both 17-estradiol and genistein significantly stimulated capacitation and acrosome loss, but the inclusion of Rp-cAMPS essentially abolished these responses; Rp-cAMPS had no detectable effect when used alone (Figure 7). These results are consistent with enhanced cAMP production in response to the xenobiotics and steroids.
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Evidence supporting those conclusions was sought by carrying out direct evaluation of cAMP production. Uncapacitated mouse sperm suspensions were prepared and treated for 2 min with 100 nmol/l genistein or 100 nmol/l nonylphenol (n = 5–6), then measured for cAMP content in the cells. CGS 21680 at 500 nmol/l served as a positive control; this is an agonist acting on stimulatory A2A adenosine receptors that has been shown to stimulate cAMP production within 2 min in uncapacitated sperm suspensions (Adeoya-Osiguwa and Fraser, 2005). The results showed that both xenobiotics and CGS 21680 significantly stimulated cAMP production compared with untreated control suspensions, responses to the xenobiotics being essentially the same as that seen with CGS 21680 (Figure 8).
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Series VII: pertussis toxin blocks responses to xenobiotics
Pertussis toxin, which irreversibly inactivates a number of inhibitory G subunits, was used to investigate whether inhibitory G proteins might be involved in responses to these xenobiotics. Uncapacitated mouse sperm suspensions were incubated for approximately 35 min in the presence of 10 nmol/l genistein and 10 nmol/l daidzein, each with/without 200 ng/ml pertussis toxin; to ensure that the pertussis toxin had time to penetrate the cells, it was added 6 min before the xenobiotics (n = 3). Consistent with other studies we have carried out (e.g. Mededovic and Fraser, 2005), pertussis toxin on its own had no detectable effect (Figure 9). However, when used in combination with genistein and daidzein, it was able to block the usual stimulatory responses obtained with these xenobiotics. This suggests the involvement of an inhibitory G-protein at some point in the response pathway.
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Series VIII: dideoxyadenosine blocks responses to xenobiotics
2',5'-Dideoxyadenosine (ddAdo), a specific inhibitor of the P-site on membrane-associated adenylyl cyclases (mACs; Johnson et al., 1997), was used to investigate whether mACs were involved in responses to xenobiotics. Uncapacitated mouse sperm suspensions were incubated for approximately 35 min in the presence of 10 nmol/l genistein with/without 100 µmol/l ddAdo (n = 3). Used on its own, ddAdo had no significant effect, again consistent with other studies using ddAdo in a similar system (Baxendale and Fraser, 2003a; Mededovic and Fraser, 2005). When used with genistein, however, ddAdo essentially blocked the responses to the xenobiotic (Figure 10). In the first replicate, 10 nmol/l daidzein was also tested and ddAdo also blocked responses to this compound; for simplicity, only genistein was used in subsequent replicates.
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Discussion |
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This study addressed a number of questions, the first being whether human spermatozoa, like their mouse counterparts, are affected by exposure to low concentrations of xenobiotics categorized as being weakly estrogenic. Genistein, the chosen test compound, was evaluated at concentrations from 1 to 100 nmol/l, the same concentrations previously tested on mouse gametes (Adeoya-Osiguwa et al., 2003
). Human spermatozoa responded to genistein in the same way as mouse spermatozoa but proved to be even more responsive, with 1 nmol/l being as effective as 100 nmol/l in accelerating capacitation and stimulating acrosome loss. In contrast, mouse spermatozoa required at least 10 nmol/l to elicit a maximal response (Figure 1A and B). In addition, there were more acrosome-reacted cells (30–37%) in the human sperm suspensions following treatment than had been seen in mouse sperm suspensions in our earlier study (17–22%; Adeoya-Osiguwa et al., 2003).
Daidzein, like genistein, is an isoflavone associated with soya and other legumes and proved to have the same effect as genistein on uncapacitated mouse spermatozoa, with responses being of a similar magnitude. This is consistent with the two compounds having structural features in common and suggests that both act in similar manner. Thus, soya-containing products have at least two components with the potential to alter sperm function. In experiments using genistein as a protein tyrosine kinase inhibitor, daidzein is often included to serve as the inactive analogue of genistein (e.g. Pukazhenthi et al., 1998). The fact that we obtained the same results with both daidzein and genistein makes it unlikely that any of the effects we have seen reflect tyrosine kinase inhibition.
Since many xenobiotics are present in the environment, both humans and animals are likely to be exposed to more than one xenobiotic at any given time, although probably each at a low concentration. The question then arises whether exposure to such combinations is more effective than exposure to any single xenobiotic. All the three xenobiotics evaluated in our earlier study (genistein, 8-prenylnaringenin and nonylphenol) show some estrogenic activity but do not share great structural similarities and their estrogenic potency is very different. Despite this, they all appear to elicit the same responses in spermatozoa (Adeoya-Osiguwa et al., 2003). Therefore, we investigated whether combinations of any two, at low concentrations that produced submaximal responses, would have a greater effect than either used individually.
The results indicate that combinations of low concentrations of these chemicals can produce greater effects than the individual compounds alone. The first combination involved genistein and nonylphenol, the latter being much less potent than genistein when used at 1 nmol/l; however, responses to the combination were significantly greater than each used individually. When genistein and 8-prenylnaringenin at both 1 and 0.1 nmol/l were combined, again the acceleration of capacitation was greater than when compounds were used singly. It is interesting to note that both compounds were almost as effective at 0.1 nmol/l as at 1 nmol/l. Thus, these xenobiotics are very potent in their action on spermatozoa, despite their relatively weak estrogenic properties when tested in standard bioassays (Routledge and Sumpter, 1996; Milligan et al., 1999). The fact that combinations were more effective than individual xenobiotics suggests that they have a common mechanism of action; thus, the combined concentration of the two effectors can elicit a greater response than either one used alone.
The inability of hydroxytamoxifen to block responses to these xenobiotics suggested that classical ERs may not be involved (Adeoya-Osiguwa et al., 2003). To explore this further, 17-estradiol, which generally binds poorly to ERs (Dykens et al., 2005), and BSA-conjugated 17-estradiol, which is too large to pass through the plasma membrane and bind to intracellular ERs, were compared with 17-estradiol for their ability to elicit responses in uncapacitated mouse spermatozoa. The fact that all three compounds significantly accelerated capacitation and acrosome loss to the same extent (Figure 5) supports our hypothesis that classical intracellular ERs are not involved in the responses.
Since all three estradiols were equally effective, we wondered whether these were nonspecific responses to steroid-like compounds or whether there might be some structure–function relationship. This was addressed by comparing 17-estradiol, 5-dihydrotestosterone and progesterone. Like 17-estradiol, 5-hydrotestosterone significantly accelerated capacitation but, unlike 17-estradiol, it did not stimulate the acrosome reaction. Progesterone had no significant effect on either parameter. Biologists interested in mechanisms involved in triggering the acrosome reaction will be aware that progesterone is often used to initiate the acrosome reaction, but it can do so only in capacitated cells (O’Toole et al., 1996). In the present study, uncapacitated mouse spermatozoa were incubated in the presence of steroid for only a short time, insufficient to allow many cells to complete capacitation, and so there were few cells that would have been able to respond to progesterone. Progesterone also failed to accelerate capacitation and acrosome loss in human sperm suspensions pre-incubated for 1 h before progesterone treatment for 30 min (Fraser and Osiguwa, 2004); in contrast, addition of progesterone to human sperm suspensions pre-incubated under capacitating conditions for 5 h did trigger the acrosome reaction in a significant proportion of the capacitated cells (e.g. O’Toole et al., 1996). These results indicate that not all steroids are equivalent in their ability to affect sperm function.
The fact that BSA-conjugated 17-estradiol was as effective as 17-estradiol suggests that the initial actions occur at the sperm plasma membrane surface, and the rapidity of response indicates nongenomic effects. The very high, nonphysiological concentrations of steroids needed to mimic the effects of low concentrations of xenobiotics are also not consistent with involvement of intracellular ERs. Studies in somatic cell systems have provided functional evidence for the presence of plasma membrane ERs mediating very rapid responses (Rønnekleiv and Kelly, 2005), but such receptors have yet to be isolated and characterized (Toran-Allerand, 2004). As discussed at some length in our earlier study (Adeoya-Osiguwa et al., 2003), attempts to determine whether mammalian spermatozoa have ER and ER, the classical ERs, have been rather inconclusive to date. A putative surface membrane-associated nongenomic ER of approximately 29 kDa, considerably smaller than ER and ER (approximately 50–65 kDa; Lambard et al., 2004), has been reported in human spermatozoa (Luconi et al., 1999), but there does not appear to have been further substantive investigation of this protein (Luconi et al., 2004).
What mechanism of action might be involved in these responses? Numerous studies have shown that cAMP plays a pivotal role in sperm physiology, with many treatments that accelerate capacitation causing an increase in cAMP. Furthermore, continuous stimulation of cAMP production appears to be associated with acrosome loss (Fraser et al., 2003). Therefore, we tested the hypothesis that these xenobiotics cause a rise in cAMP that then elicits the observed responses. Inclusion of the protein kinase-A inhibitor Rp-cAMPS completely blocked responses to both 17-estradiol and genistein, consistent with xenobiotic stimulation, causing an increase in cAMP generation. Direct confirmation that this is the case was obtained by measuring cAMP production in response to genistein and nonylphenol; both xenobiotics significantly stimulated cAMP within 2 min, and the magnitude of the response was very similar to that obtained with CGS 21680, a stimulatory adenosine receptor agonist known to stimulate cAMP production in spermatozoa (Adeoya-Osiguwa and Fraser, 2005).
The possible involvement of G proteins in the observed responses was investigated by using pertussis toxin, which can bind to and inactivate a number of inhibitory G subunits. Although pertussis toxin itself had no detectable effect (Figure 9), it was able to block responses to both genistein and daidzein, suggesting that inhibitory G proteins are involved. We have shown that mammalian spermatozoa have a number of inhibitory G subunits (Fraser and Adeoya-Osiguwa, 1999; Baxendale and Fraser, 2003b). However, it seems unlikely that the xenobiotics are acting directly on mACs, since most mACs are usually stimulated by the stimulatory Gs. In a recent study, we found evidence for the presence of several mAC isoforms in mammalian spermatozoa, with mAC2, mAC3 and mAC8 being most abundant and mAC1 and mAC4 being less abundant (Baxendale and Fraser, 2003a). Although some isoforms can be stimulated by G
rather than G, the former appears to have no direct effect on mAC3, mAC8 and mAC9 and can only activate mAC2 and mAC4 in the presence of activated Gs (Defer et al., 2000). Therefore, it seems probable that the inhibitory G proteins are acting on a different target.
In various somatic cell systems, rapid responses to estradiol have been shown to involve an increase in the intracellular Ca2+ concentration and generation of cyclic nucleotides (Kelly and Levin, 2001). An increase in Ca2+ in response to xenobiotics could have a stimulatory effect on mACs and/or the recently identified soluble adenylyl cyclase (sAC), both of which are present in spermatozoa and can be stimulated by Ca2+ (mACs, Defer et al., 2000; sAC, Jaiswal and Conti, 2003; Litvin et al., 2003). The mechanism whereby intracellular Ca2+ might be increased is not clear, although it is unlikely to involve Ca2+ channels since these function primarily in capacitated spermatozoa, during initiation of the acrosome reaction (O’Toole et al., 1996). One possibility is that the inhibitory G proteins are inhibiting a Ca2+ extrusion mechanism such as Ca2+-ATPase or a Na+/Ca2+ exchanger. A similar mechanism involving inhibitory proteins has been suggested for angiotensin II and its G-protein-coupled receptor (Mededovic and Fraser, 2005).
To try to differentiate between responses involving mACs and sAC, we used 2'5'-deoxyadenosine (ddAdo) in combination with genistein and daidzein. The sAC is reported to be insensitive to both G proteins and other molecules that are known to act on mACs, including ddAdo which acts on the P-site of mACs, a region not present on sAC (Buck et al., 1999). The fact that ddAdo essentially blocked the usual responses to these xenobiotics would suggest that much of the cAMP generated comes from mAC activation rather than sAC activation.
The ability of genistein to cause 30% of human spermatozoa to both capacitate and undergo the acrosome reaction within 1 h is very striking. In our experience, human spermatozoa do not normally undergo spontaneous acrosome reactions readily, even when incubated for 24 h. After 1 h of incubation under capacitating conditions, human sperm suspensions typically have <10% acrosome-reacted cells (see Figure 1 and Fraser and Osiguwa, 2004), and in a study where suspensions were incubated for 24 h, only about 20% of cells had acrosome-reacted spontaneously (DasGupta et al., 1994).
Human spermatozoa show the same responses in vitro to genistein as do mouse spermatozoa; if such responses were to occur in vivo, it is possible that this could have a significant impact on sperm fertilizing ability. This is because already acrosome-reacted spermatozoa have lost the plasma membrane over the anterior part of the sperm head and so are unable to bind to the zona pellucida, a necessary prerequisite for successful fertilization (de Lamirande et al., 1997). While a recent study of men given daily supplements of genistein for 2 months found no significant effect on semen quality (Mitchell et al., 2001), the parameters assessed (semen volume, sperm concentration, motility and morphology) do not reflect sperm fertility per se. The low nanomolar concentrations of xenobiotics used in the present study elicited subtle and significant effects on both human and mouse sperm function, but there is no reason to expect these low concentrations to produce any deleterious effects on testicular function or commonly measured semen parameters.
It is likely that spermatozoa encounter, simultaneously, several xenobiotics at nanomolar concentrations in vivo within the female reproductive tract. Mitchell et al. (2001) found a mean value of 1.0 µmol/l in the plasma of their volunteers taking genistein daily. Furthermore, plasma levels of approximately 100 nmol/l free genistein and approximately 500 nmol/l free daidzein have been found in women within 3 h of their consuming 25 g soy milk (Zhang et al., 2003), and high nanomolar levels of individual flavonoids can be found in humans with high vegetable diets (e.g. hesperetin: 325 nmol/l; naringenin: approximately 110 nmol/l; Radtke et al., 2002). All these are well within the concentration range that we found to affect spermatozoa. Regarding animals, soya is the major protein source found in most commercially available natural-ingredient diets for laboratory animals; it is also found in many feeds used for agricultural animals (Lephart et al., 2004) and animals maintained in zoos and similar establishments (A. Hartley, personal communication). While a number of studies have examined the effects of dietary soya or genistein on reproductive parameters in male and female rodents, these have largely focused on the long-term effects of soya exposure during development (e.g. Atanassova et al., 2000; Delclos et al., 2001). Other studies have examined the effects of soya isoflavones administered to adults for long periods (e.g. Gallo et al., 1999; Faqi et al., 2004) or occasionally in multigeneration studies (McVey et al., 2004). There has been a general lack of consistency in the findings of such studies, probably reflecting the variety of experimental protocols involved and end-points assessed. In the current context, it is sufficient to emphasize that many of the end-points in previous studies have been based on morphological assessments of gonadal or gamete structure rather functional end-points. As such, even negative results do not preclude the possibility that xenobiotics within the female tract could affect the physiology of individual spermatozoa and their fertilizing capacity.
In light of current concerns about the links between the exposure to environmental chemicals and possible long-term effects on fertility, further investigations are required to determine the significance and mechanisms underlying the very acute action of such environmental compounds on the key processes of capacitation and the acrosome reaction in spermatozoa of both humans and other mammals.
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