Prostaglandins induce calcium influx in human ... - Semantic Scholar

Molecular Human Reproduction vol.4 no.6 pp. 555–561, 1998

Prostaglandins induce calcium influx in human spermatozoa

Yasufumi Shimizu1, Ayumi Yorimitsu1, Yoshinori Maruyama1, Toshiro Kubota1, Takeshi Aso1 and Richard A.Bronson2,3 1Department of Obstetrics and Gynecology, Faculty of Medicine, Tokyo Medical and Dental University, Tokyo 113, Japan, and 2Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology, State University of New York at Stony Brook, Stony Brook, NY 11794–8091,USA 3To

whom correspondence should be addressed

Progesterone, prostaglandin and follicular fluid are reported to enhance the acrosome reaction through the influx of extracellular calcium into the cytoplasm of human spermatozoa. Prostaglandins are present within the male reproductive tract, and high concentrations of prostaglandins exist in seminal fluid. In order to investigate the mechanisms by which prostaglandins enhance the acrosome reaction through calcium influx, the intracellular calcium response induced by progesterone, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2) and follicular fluid was measured using fura-2. PGE1 and PGE2 promoted calcium influx dose dependently through dihydropyridine insensitive calcium channels. Refractoriness of the elevation of intracellular Ca21 concentration ([Ca21]i) to a second stimulus occurred when 60 µg/ml PGE1 was administered 100 s after the prior administration of 60 µg/ml of PGE1, and similarly when 1 µg/ml of progesterone was administered 100 s after the prior administration of 1 µg/ml of progesterone. Refractoriness also occurred when 60 µg/ml PGE1 was administered after the prior addition of 60 µg/ml PGE2, but did not occur between PGE1 and progesterone. Pertussis toxin (PTX) did not modify the changes in [Ca21]i after the addition of PGE1 or PGE2. In conclusion, PGE1 and PGE2 promoted calcium influx through PTX-insensitive calcium channels which appeared to be recognized by a common receptor different from that of progesterone. Key words: calcium/calcium channel/progesterone/prostaglandin/spermatozoa

Introduction It is well known that a large influx of extracellular calcium triggers the acrosome reaction (Yanagimachi, 1994). Recent studies have shown that progesterone and follicular fluid cause an immediate increase in calcium in both non-capacitated and capacitated human spermatozoa, and progesterone may stimulate the acrosome reaction in human spermatozoa (Margalioth et al., 1988; Thomas and Meizel, 1988; Osman et al., 1989; Blackmore et al., 1990; Baldi et al., 1991). We reported that the steady state intracellular calcium concentration ([Ca21]i) of human spermatozoa was increased following in-vitro capacitation, as was the peak [Ca21]i evoked by progesterone (Shimizu et al., 1993). Prostaglandins are involved in the male reproductive tract, and high concentrations of prostaglandins are reported to exist in seminal fluid (Templeton et al., 1978) and cervical mucus (Charbonnel et al., 1982). As spermatozoa gain their fertilizing ability during their epididymal transit, the possibility exists that prostaglandins influence the sperm fertilizing ability during this period. It was reported that the coordinated loss of specific factors originating from epididymal fluid or seminal plasma is associated with extensive reorganization of membranes and initiation of signal transduction mechanisms that cause spermatozoa to beome capacitated (Lamirande et al., 1997). Meizel and Turner (1983, 1984) reported that arachidonic acid, prostaglandin E2 (PGE2) and 5- and 12-hydroxyeicosatetraenoic © European Society for Human Reproduction and Embryology

acids (5-HPETE and 12-HPETE) stimulate the acrosome reaction of hamster spermatozoa. Prostaglandins are reported to enhance sperm motility (Aitken and Kelly, 1985), and the penetration of human spermatozoa into zona-free hamster oocytes, as well as to increase the [Ca21]i of human spermatozoa measured using quin-2 (Aitken et al., 1986). Also, it was reported that PGE2 increased the acrosome reaction of guinea pig spermatozoa initiated by Ca21 (Joyce et al., 1987). Human spermatozoa have been shown to synthesize prostaglandins through the existence of cyclo-oxygenase activity (Roy and Ratnam, 1992). Also, in a study by Shalev et al. (1994), bovine spermatozoa synthesized prostaglandins in the presence of arachidonic acid or melittin (phospholipase A2 activator). Shalev et al. (1994) also reported that PGE2, but not prostaglandin F2α (PGF2α) increased the Ca21 uptake linearly during the first 10 min of incubation. In bovine spermatozoa, cyclo-oxygenase is localized mainly in the sperm head (Shalev et al., 1994). Exogenous PGE2 enhances Ca21 uptake and stimulates the acrosome reaction, which is completely inhibited by the lipoxygenase inhibitor, indicating that the lipoxygenase pathway is involved in the mechanisms by which the cyclo-oxygenase pathway stimulates the acrosome reaction (Breitbart and Spungin, 1997). Tesarik et al. (1993) reported that different signal transduction mechanisms exist between progesterone- and zona pellucida-induced acrosome reaction. We further investigated whether prostaglandin555

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induced calcium influx was mediated through different pathways from that of progesterone. In this study, we demonstrate that prostaglandin E1 (PGE1) and PGE2 promote an influx of extracellular calcium in a concentration-dependent manner through a common pathway by mechanisms different from those associated with progesterone-induced calcium responses. Dihydropyridines did not abrogate this response.

Materials and methods Preparation of human spermatozoa Semen samples were obtained from ejaculates of known fertile donors and from those of infertile men submitted for semen analysis to the Department of Obstetrics and Gynecology, Tokyo Medical and Dental University Hospital, Japan. Semen was evaluated following the World Health Organization criteria (WHO, 1992). After specimens were allowed to liquefy at room temperature for at least 30 min, a highly motile population was recovered after 60 min swim-up in Biggers– Whitten–Whittingham (BWW) medium supplemented with 5 mg/ml human serum albumin (HSA). For swim-up, 500 µl aliquots of semen were injected under 2 ml BWW/HSA 5 mg/ml in 15 ml loosely capped conical tubes. After a 60 min incubation at 37°C in 5% CO2 in air, the upper 1–1.5 ml of medium was collected, and spermatozoa were washed by centrifugation for 8 min at 600 g. Thereafter spermatozoa were resuspended in BWW/HSA 30 mg/ml, and incubated at 37°C in 5%CO2 for overnight (18–22 h) for capacitation. These spermatozoa were used within 10 min of their removal from the incubator for [Ca21]i measurement. Human follicular fluid was obtained at the time of oocyte retrieval, as part of the in-vitro fertilization (IVF)/embryo transfer programme in Tokyo Medical and Dental University Hospital, with patient’s permission. Follicular fluid was centrifuged for 15 min at 800 g, and the supernatant was stored frozen at –20°C until use. Measurement of changes in cytosolic free Ca21 concentration Changes in cytosolic free Ca21 concentration, [Ca21]i, were monitored fluorimetrically using the Ca21-sensitive indicator fura-2AM as previously described (Thomas and Meizel, 1988; Blackmore et al., 1990; Shimizu et al., 1993). Fura-2 and its Ca21 complex fluoresce strongly but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca21bound dye to free dye and hence cytosolic free calcium (Tsien et al., 1985). Briefly, capacitated spermatozoa (7.53106/ml) were loaded with 2 µM fura-2AM for 45 min at 37°C. Loaded spermatozoa were rinsed and assayed in BWW/HSA 30 mg/ml. Fluorescence measurements were performed on a Hitachi spectrofluorometer F2000 (Tokyo, Japan) at excitation wavelengths of 340 and 380 nm and emission wavelength 510 nm with slit widths set at 5 nm. An aliquot of fura-2 loaded spermatozoa was introduced into a 4 ml cuvette, and continually stirred in a thermostatic cuvette holder (37°C). Solutions containing test compounds were added to the cuvette at a 1:100 dilution. At 60 s after data collection was started, progesterone was dissolved in dimethlysulphoxide (DMSO) at a concentration of 100 µg/ml, and 15 µl was added to the cuvette containing 1.5 ml sperm suspension to make a final concentration of progesterone 1 µg/ml. PGE1, PGE2, and PGF2α were dissolved in DMSO at an concentration of 6 mg/ml, 600 µg/ml and 60 µg/ml. The ratio 340:380 was converted to [Ca21]i according to the formula described by Gynkiewicz et al. (1985). Rmax was the ratio 340:380 when intracellular calcium was saturated, and Rmin was the ratio

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340:380 when intracellular calcium was depleted. Rmax and Rmin were determined in the presence of 0.01% (wt/vol) digitonin and 15 mM ethylene-bis EGTA respectively, as previously described (Thomas and Meizel, 1988; Blackmore et al., 1990). After the incubation with 100 ng/ml Pertussis toxin (PTX) for 3 h, [Ca21]i induced by PGE1 or PGE2 were compared with the [Ca21]i in the absence of PTX.

Reagents Fura-2AM was purchased from Molecular Probes (Junction City, OR, USA) and dissolved in DMSO and stored at 0°C until use. Bovine serum albumin (BSA), HSA, penicillin G, streptomycin sulphate, progesterone, PGE1, PGE2, PGF2α and ethylene-bis (oxy-ethylenenitriolo)tetraacetic acid were purchased from Sigma Chemical Company (St Louis, MO, USA). PTX was purchased from Calbiochem (San Diego, CA, USA). Progesterone and prostaglandins were each dissolved in DMSO. Digitonin and pertussis toxin were dissolved in distilled water. Ethylene-bis (oxy-ethylenenitriolo) tetraacetic acid was dissolved in 1 M Tris (pH 8.3). Statistical analysis Results were expressed as the mean 6 SD. Statistical comparisons were made using Student’s t-test for paired or non-paired samples. Correlations were calculated using Pearson’s correlation coefficient.

Results The response of capacitated human spermatozoa to challenge with progesterone, PGE1, PGE2 and PGF2α and changes in [Ca21]i of capacitated spermatozoa under steady-state and agonist-challenge conditions were monitored fluorometrically as described in the Materials and methods section. Cells challenged with PGE1 or PGE2 responded with a prompt increase in [Ca21]i. Challenge of spermatozoa with 60 µg/ml PGE1 resulted in a brisk increment in [Ca21]i that rapidly returned to a new, elevated, steady-state plateau value (Figure 2A). Increase of [Ca21]i from baseline following exposure to 1 µg/ml progesterone was 203 6 135% (n 5 19, Figure 1A), not significantly different from 60 µg/ml PGE1 (264 6 241%; n 5 8, Figure 2A). Challenge of spermatozoa with 60 µg/ml PGE2 also resulted in a brisk increment in [Ca21]i , but the increase of [Ca21]i in response to PGE2 was 119 6 65% (n 5 13), lower than 60 µg/ml PGE1 (P , 0.05, Figure 1C). Increment of [Ca21]i by PGE1 was dose-dependent (Figure 2A,B,C), and there was no increase of [Ca21]i when 600 ng/ ml PGE1 was administered (Figure 2C). The increment of [Ca21]i induced by 60 µg/ml PGE1 was diminished following the prior administration of 600 ng/ml PGE1 (Figure 2C), indicating the refractoriness of [Ca21]i increase by PGE1. Refractoriness also occurred with the prior administration of the same amount of PGE1. Thus, the increase of [Ca21]i induced by the addition of 60 µg/ml PGE1 100 s after the prior administration of 60 µg/ml PGE1 was 27 6 31%, which was smaller than the increase of [Ca21]i induced by 60 µg/ml PGE1 without prior addition of 60 µg/ml PGE1 (264 6 241%, n 5 5, P , 0.05). This suggested that the receptor of PGE1 was desensitized. Refractoriness to PGE1 occurred following a prior challenge with PGE2 as well (data not shown). The deletion of Ca21 from assay buffer with the administration of 3 mM EGTA abrogated the prostaglandin-induced Ca21


Figure 1. Refractoriness of sperm response to prostaglandin E1 (PGE1) is independent of response to progesterone. Progesterone 1 µg/ml was administered 100 s after the prior addition of (A) 1 µg/ml progesterone or (B) PGE1 60 µg/ml. The increment of intracellular calcium concentrations ([Ca21]i) by 60 µg/ml PGE1 was reduced when administered 100 s after the prior addition of (C) PGE2 60 µg/ml. Representative tracings of [Ca21]i in capacitated human spermatozoa in response to (D) PGE1 60 µg/ml or (E) progesterone 1 µg/ml. (F) PGE1 60 µg/ml and progesterone 1 µg/ml was administered simultaneously.

Figure 2. Effect of prostaglandin E1 (PGE1) on sperm cytosolic free calcium. Representative tracings of the response of intracellular calcium concentrations ([Ca21]i) in response to (A) PGE1 60 µg/ml, (B) PGE1 6 µg/ml, and (C) PGE1 0.6 µg/ml in capacitated human spermatozoa from known fertile donors. PGE1 increased [Ca21]i in a dose-dependent manner. (E) 10–6 M nicardipine did not completely block the increment of [Ca21]i by PGE1 60 µg/ml. (F) The response of human spermatozoa to PGE1 60 µg/ml was abrogated by the chelation of extracellular calcium with 3 mM EGTA. (D) is included as the control for E and F. Different proven fertile donor spermatozoa were utilized in A–C and D–F.

increment (Figure 2F), but dihydropyridines did not completely block the response to 60 µg/ml PGE1 (Figure 2E, compared with Figure 2D). Refractoriness of the increment of [Ca21]i arose when 1 µg/ml progesterone was administered (33 6 62%, n 5 7, P , 0.01) 100 s after the prior addition of 1 µg/ml progesterone (Figure 1A). Refractoriness did not occur when 1 µg/ml progesterone was administered (302 6 251%, n 5 13, P 5 0.32) 100 s after the addition of 60 µg/ml PGE1 (Figure 1B), suggesting that PGE1 and progesterone promote calcium influx through different pathways. The increment of [Ca21]i induced by 60 µg/

ml PGE1 was reduced when administered 100 s after the addition of 60 µg/ml PGE2 (Figure 1C), suggesting that a common pathway exists in the mechanism through PGE1 and PGE2. Increase of [Ca21]i following simultaneous administration of PGE1 and progesterone was 1183 6 87% (n 5 3, Figure 1F), higher than PGE1 alone (P , 0.01, Figure 1E), suggesting an additive effect exists between PGE1 and progesterone. PGF2α (60 µg/ml) did not increase [Ca21]i of human spermatozoa, and PGF2α did not modify the changes of [Ca21]i after the addition of 60 µg/ml PGE1 or 1% human follicular fluid (Figure 3). 557

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(Anderson et al., 1994), but there was no increase of [Ca21]i of human spermatozoa when administered up to 10–6 M (Figure 6B,C). Proven fertile donor spermatozoa were used for the experiments in Figures 1, 2, 3, 5 and 6. In Figure 4, spermatozoa from two proven fertile donors and six infertile patients were used. With regard to the semen from six infertile men, four ejaculates were normal by World Health Organization criteria (WHO, 1992). In one ejaculate, initial sperm motility was 30% and 40% in the other, but sperm morphology was normal in both. Figure 3. Effect of administration of prostaglandin F12α (PGF2α) 60 µg/ml on intracellular calcium concentrations ([Ca21]i). Prior administration of PGF2α 60 µg/ml resulted in no inhibition or enhancement of the increment of [Ca21]i caused by PGE1 60 µg/ml or 1% follicular fluid. Control data, i.e. responses induced by 60 µg/ml PGE1 or 1% follicular fluid alone are not shown because they were almost the same as those shown in the figure.

Figure 4. Correlation curve between percentage increase of intracellular calcium concentrations ([Ca21]i) by progesterone 1 µg/ml and prostaglandin E1 (PGE1) 60 µg/ml, when measured simultaneously in eight different donors and infertile men.

The percentage increase of [Ca21]i following challenge with 60 µg/ml PGE1 was positively correlated with that induced by 1 µg/ml progesterone when measured simultaneously in eight different donors and infertile men (r 5 0.793, P , 0.019, Figure 4). After the incubation with 100 ng/ml PTX for 3 h, increase of [Ca21]i from baseline induced by 60 µg/ml PGE1 was 86 6 22% (n 5 3, Figure 5A), not different from that without PTX (101 6 85%; n 5 3; Figure 5B). It was the same with PGE2, as the increase of [Ca21]i from baseline was 98 6 99% (n 5 3) with 100 ng/ml PTX (Figure 5C), and 83 6 89% (n 5 3) without PTX (Figure 5D). Also, the increment of [Ca21]i induced by 1% human follicular fluid was not found to be modified on comparison of the changes with PTX (Figure 5B,D). Pentoxyfylline, a phosphodiesterase inhibitor which increases intracellular cyclic AMP (cAMP) concentration in spermatozoa, was added up to a concentration of 1 mg/ml, and there was no increase of [Ca21]i (Figure 6A). Angiotensin II and endothelin-1, which both increase [Ca21]i in endothelial cells, had no effects in increasing [Ca21]i of spermatozoa. We also tested atrial natriuretic peptide which was reported to stimulate the acrosome reaction of human spermatozoa 558

Discussion In the present experiments, PGE1 and PGE2 promoted calcium influx through dihydropyridine insensitive calcium channels which are different from those of progesterone, while PGE1 and PGE2 appeared to be recognized by a common receptor. PGE1 was more potent in increasing [Ca21]i than PGE2. PGF2α was without effect. In our experiments, responses of [Ca21]i varied between individuals and even with the same individual when tested on different days. This variation was responsible for the high SD associated with the percentage increases in [Ca21]i reported in the results. Since the culture medium used was not altered between assays, the high variablity suggests that sperm function varies between ejaculates. It has been suggested that the influx of extracellular calcium triggers the acrosome reaction of human spermatozoa. Human follicular fluid enhances sperm fertilizing ability by increasing [Ca21]i. Human follicular fluid contains both progesterone and prostaglandins, which increase [Ca21]i of human spermatozoa and enhance sperm fertilizing ability (Aitken et al., 1986; Blackmore et al., 1990; Amiel et al., 1991; Shimizu et al., 1993; Krausz et al., 1994). In this study we demonstrated that prostaglandins promoted the influx of extracellular calcium through a different calcium channel from that of progesterone, and that the effects of PGE1 and progesterone was additive. Given these results, we speculate that the prostaglandin receptor in the sperm plasma membrane is separate from the progesterone binding site. We have previously reported that the [Ca21]i increase induced by 1% human follicular fluid is much greater than that of 1 µg/ ml progesterone or 60 µg/ml PGE1 (data not shown). Hence, there arises the possibility that the [Ca21]i increase following the exposure of spermatozoa to human follicular fluid is the accumulation of the [Ca21]i increase induced by progesterone and prostaglandins, although other factors contained in human follicular fluid cannot be excluded. As the increment of [Ca21]i induced by PGE1 was correlated with that induced by progesterone, there may be a common mechanism to induce calcium influx by agonists within fertile and infertile men; the amount of calcium influx revealing their fertilizing ability. Aitken et al. (1986) reported that exposure to PGE1 and PGE2, but not PGF2α, induced a significant rise in the values of cytoplasmic Ca21. Stimulation of oocyte penetration with PGE1 and PGE2 was significantly enhanced when these compounds were presented to the spermatozoa in medium with


Figure 5. Pertussis toxin (PTX) was without effect on sperm responsiveness. Representative tracings of the intracellular calcium concentration ([Ca21]i) response to (A) prostaglandin E1 (PGE1) 60 µg/ml, or (C) PGE2 60 µg/ml in the presence of 100 ng/ml PTX for 3 h in capacitated human spermatozoa compared with its absence (B,D).

Figure 6. (A) 1 mg/ml pentoxyfylline, (B) 10–5 M angiotensin II, and (C) 10–6 M atrial natriuretic peptide (ANP) and 10–6 M endothelin-1 (ET-1) had no effect on capacitated spermatozoa; however, the spermatozoa were responsive to progesterone 1 µg/ml.

high osmolarity. Our data confirm their result, although quin2, a different indicator dye, was used to measure [Ca21]i. Aitken et al. (1986) also reported that voltage-sensitive calcium channels do not appear to be involved, since verapamil did not significantly depress the penetration rate enhanced by PGE1. Our result agreed with their study, as dihydropyridines, voltage-sensitive L-type calcium channel blockers, did not block the increase of [Ca21]i by PGE1. We previously reported

that dihydropyridines did not block the increase of [Ca21]i by progesterone (Shimizu et al., 1993). PGE1 has been reported to act as a calcium ionophore (Kirtland and Baum, 1972). Aitken et al. (1985, 1986) suggested that prostaglandins might mediate calcium uptake of spermatozoa via an ionophore-like effect. Our results, however, suggest that the prostaglandin effect on human spermatozoa is mediated by specific receptors. We noted differences in response of spermatozoa to PGE and PGF, as well as refractoriness of spermatozoa to stimulus with PGE1 and PGE2. Three different mouse PGE receptors (EP1, EP2, EP3) have been cloned using the mouse thromboxin A2 receptor cDNA as a probe. These clones all specifically bind PGE2 and each generates a different signal, indicating that they are all PGE receptors. Ligation of the EP3 receptor has been associated with increases in intracellular calcium (Nariyama, 1994). Although a PGF receptor has also been identified, the failure of PGF2α to promote calcium uptake by capacitated spermatozoa argues against its presence in human spermatozoa. Recently, Aitken et al. (1996) reported that down-regulation of progesterone receptor occurred in the extragenomic action of progesterone on human spermatozoa. Our study suggests that similar down-regulation mechanisms of sperm surface prostaglandin receptor exist. Refractoriness of response to PGE1, PGE2 and also between PGE1 and PGE2, was noted as well but did not occur between PGE and progesterone, again suggesting different pathways of promotion of calcium uptake. The dose of prostaglandins used in this study is physiological as the concentration of prostaglandins in human seminal plasma was reported to be .1 mg/ml, largely due to the presence of considerable quantities of PGE (Samuelsson, 1963). Human cervical mucus was also reported to contain large amounts of prostaglandins (Charbonnel et al., 1982). Mack et al. (1992) reported that arachidonic acid, PGE1 and prostacyclin (PGI2) induced the acrosome reaction when added to the capacitated spermatozoa, and that the arachidonic acid559

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induced acrosome reaction was dependent upon extracellular calcium. Our results agree with theirs in that calcium influx by prostaglandins was dependent on extracellular calcium, as deletion of Ca21 from assay buffer with EDTA abrogated the prostaglandin-induced response. This finding indicates that influx of extracellular calcium into the cell through calcium channels, whether mediated by progesterone (Blackmore et al., 1990) or prostaglandins, is the primary route of alternations in intracellular calcium concentration in human spermatozoa. Aboolian et al. (1989) reported that PGE1 and PGE2 but not PGF2α mobilized Ca21 from intracellular stores in a concentration-dependent manner in Madin-Darby canine kidney (MDCK) cells. Although, there is a difference in the pathway to increase [Ca21]i, as mobilization of Ca21 is from intracellular stores in MDCK cells and influx from extracellular medium in human spermatozoa, PGE1 and PGE2 appeared to interact with a common receptor, a finding similar to our own. The treatment of human spermatozoa with PTX, an inhibitor of G protein mediated events, did not modify the changes of [Ca21]i after the administration of PGE1 and PGE2. Although PTX has been shown to inhibit the acrosome reaction induced by zona pellucida in mouse (Endo et al., 1987) and human (Lee et al., 1992) spermatozoa, Tesarik et al. (1993) reported PTX did not modify the changes in the intracellular concentration of calcium ions occurring after progesterone addition. As the increment of [Ca21]i by PGE1 and PGE2 were also not affected with the incubation of PTX, signal transduction mechanisms which induce the acrosome reaction are different between zona pellucida and prostaglandins. We also compared the effect of overnight incubation of spermatozoa in capacitating medium on the increment of [Ca21]i induced by prostaglandins. There was no difference (data not shown), between the increment of [Ca21]i induced by 60 µg/ml PGE1 in fresh or in spermatozoa incubated overnight, suggesting that the prostaglandins do not appear to require capacitation to promote calcium uptake by spermatozoa. In contrast, the calcium increments of human spermatozoa in response to progesterone increased following their capacitation (Shimizu et al., 1993). Hence, progesterone appears to act via a receptor whose function is dependent upon capacitation, while the PGE receptor expression may not vary with the functional status of human spermatozoa. In conclusion, this study reinforces the notion that there are different pathways which appear independently to mediate calcium entry within the spermatozoon, and that the prostaglandins are involved in this process.

Acknowledgements This work was supported by Grant-in-Aid (07671769) from the Ministry of Education, Science and Culture of Japan.

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