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Gamete Biology |
Department of Medicine, University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada H1T 2M4
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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acrosome reaction, male reproductive tract, progesterone, sperm capacitation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The bovine seminal plasma (BSP) contains a group of acidic proteins (BSP-A1, -A2, -A3, and -30-kDa), secreted by the seminal vesicles, which constitute the major protein fraction of bovine seminal fluid (20–40 mg/ml) [21–23]. BSP-A1 and BSP-A2 have an identical amino acid sequence, but their difference resides in the degree of glycosylation; they are thus considered as a single chemical entity named BSP-A1/-A2. Upon ejaculation, the BSP proteins interact with the choline phospholipids on the sperm membrane [24]. Previous studies have shown that the BSP proteins accelerate the capacitation of bovine epididymal sperm induced by heparin and high-density lipoprotein (HDL) [25–27]. In our recent work, we showed that during the brief exposure of sperm to seminal plasma (at ejaculation), the BSP proteins alone could stimulate a significant sperm membrane cholesterol efflux accompanied by some phospholipid efflux [28, 29]. We also showed that a continuous exposure of sperm to BSP-A1/-A2 proteins (120 µg/ml) led to 30%–33% of cholesterol efflux, and this resulted in a 2-fold higher capacitation than that observed in the control sample [28]. Therefore, BSP proteins alone would appear to be sufficient to capacitate bovine epididymal sperm when incubated with them for longer periods, but this situation is not physiological because seminal plasma, which contains BSP proteins, is gradually diluted and lost during sperm transit through the female genital tract. Therefore, the sperm membrane lipid efflux induced by the BSP proteins during ejaculation prime sperm to undergo capacitation, which is then completed in the oviduct by the action of HDL (second cholesterol efflux) or heparin-like glycoaminoglycans [28].
Our results, using fibroblasts, have shown that P increased the cholesterol efflux potential of the BSP-A1/-A2 proteins [30]. Thus, the first objective of this study was to evaluate whether P also stimulates sperm cholesterol efflux induced by BSP-A1/-A2 proteins. The second objective was to determine the effect of P on bovine sperm capacitation and AR.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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BSA (fraction V fatty acid free), heparin (purified from porcine intestinal mucosa), taurine, L-epinephrine, erythrosin B, flavianic acid (naphthol), P, and lysophosphatidylcholine (lyso-PC; purified from egg yolk) were from Sigma (St. Louis, MO); penicillin G and streptomycin sulfate were from Gibco (Burlington, ON, Canada), and [3H]cholesterol (specific activity, 43.5 Ci/mmol) was from DuPont Canada (Mississauga, ON, Canada). All other chemicals used were of analytical grade and obtained from commercial suppliers.
Bovine testes and epididymides were obtained from Abattoir Les Cèdres (St. Lazare, PQ, Canada), and freshly ejaculated bovine semen collected with an artificial vagina was obtained from the Centre d'Insémination Artificielle du Québec (St. Hyacinthe, PQ, Canada). BSP-A1/-A2 proteins were isolated using gelatin-agarose affinity chromatography [23]. The adsorbed fractions were eluted and resolved on Sephadex G-75 and G-200 columns under conditions described previously [22]. The purity of the BSP-A1/-A2 proteins was confirmed by SDS-PAGE.
Sperm Labeling and Cholesterol Efflux of Epididymal Sperm
The medium used for washing and incubation of sperm was a modified Tyrodes medium, designated mTALP [25]. Caudal sperm were collected from the epididymides obtained from four different bulls, pooled, and washed three times (350 x g, 10 min) with 10 vol of mTALP. The sperm were then labeled using the method described by Langlais et al. [31] with minor modifications [28]. Studies of sperm cholesterol efflux were done by incubating the labeled ([3H]cholesterol) sperm suspension (final concentration of 5 x 107 cells/ml) with medium or medium containing BSP-A1/-A2 proteins (40 µg/ml) with or without ethanol (final concentration of 0.1%), in the presence of different concentrations of P (0–50 µg/ml) in triplicate. P was dissolved in ethanol and serially diluted. The maximum concentration of ethanol in the incubation media did not exceed 0.1%. The sperm were then incubated for 6 h at 39°C under 5% CO2, during which two samples of 50 µl were withdrawn at different times to determine the percentage of [3H]cholesterol taken up by the incubation medium. To measure the total radioactivity associated with the sperm and the suspension, 50 µl of the sperm suspension were incubated 5 min (37°C) with 200 µl of NaOH (1N) and 50 µl of dithiotreitol (5 x 10-3 M). Then 150 µl of 1% SDS were added, samples were vortexed and diluted to 1 ml with water. One hundred microliters of the final suspension were counted (Wallac liquid scintillation ß-counter, Perkin Elmer, Boston, MA). To measure the radioactivity retrieved in the incubation medium, 950 µl of mTALP were added to 50 µl of the sperm suspension and centrifuged (940 x g, 10 min). The radioactivity in 100 µl of the supernatant was determined. The percentage of [3H] cholesterol released into the medium was calculated from the radioactivity retrieved in the incubation medium divided by the total radioactivity associated with the sperm in the suspension. The experiments were repeated at least three times.
Sperm Capacitation and AR of Epididymal Sperm
To determine the number of capacitated sperm and acrosome-reacted sperm during the efflux study, aliquots of sperm were withdrawn at different times. To measure capacitation, lyso-PC was added at 100 µg/ml, and the sperm were reincubated for an additional 15 min and smeared on slide. This concentration of lyso-PC was previously shown to induce the AR in capacitated sperm but having no effect on noncapacitated sperm [32]. The lyso-PC-induced AR is a well-characterized method that has been correlated with in vitro fertilization rates and validated by electron micrograph studies [32]. Therefore, the percentage of AR induced by lyso-PC obtained included the number of capacitated sperm that were acrosome reacted in contact with lyso-PC, the number of sperm that were spontaneously acrosome reacted, and the number of capacitated sperm acrosome reacted by the treatment (for example addition of progesterone). To determine the AR, the sperm were reincubated 15 min with medium alone and smeared on slide (without addition of lyso-PC). The percentage of AR (AR noninduced by lyso-PC) included the number of sperm, which were spontaneous acrosome reacted, and the number of capacitated sperm, which were acrosome-reacted by the treatment (addition of progesterone). All these experiments were performed three times in duplicate.
Sperm Capacitation and AR of Ejaculated Sperm
Freshly ejaculated bovine sperm were washed three times (375 x g, 10 min) with mTALP and were adjusted to a concentration of 1 x 108 cells/ml. Then sperm were incubated for 6 h at a final concentration of 5 x 107 cells/ml under various conditions: with or without heparin (12 µg/ml) or with different concentrations of P (0.01–10 µg/ml) serially diluted in ethanol. The final volume of ethanol in the incubation media did not exceed 0.1%. Aliquots of each sample were taken at different times and reincubated 15 min in the presence or absence of 100 µg/ml of lyso-PC to determine the percentage of sperm that undergo the AR and the percentage of sperm that undergo the AR induced by lyso-PC (see above). In another set of experiments, washed ejaculated sperm were incubated for 6 h at a final concentration of 5 x 107 cells/ml with heparin (12 µg/ml). Aliquots of each sample were taken at different times and reincubated 0–15 min in the presence or absence of 100 µg/ml of lyso-PC or with different concentrations of P (0.5–2 µg/ml) diluted in ethanol. Then the percentage of sperm that undergo the AR was determined for each sample. All these experiments were performed three times in duplicate.
Staining Procedure
Prior to drying and staining, randomly selected slides were examined using light microscopy to verify sperm motility. The percentage of sperm that were acrosome reacted was determined on air-dried sperm smears with a naphthol yellow-erythrosin B-staining procedure [33]. For each slide, 400 spermatozoa were hand counted under light microscope (x1000, Leitz, Wetzlar, Germany). The sperm viability was estimated by the staining protocol of Dott and Foster [34]. Briefly, an aliquot of each sperm suspension (10 µl) was applied on a microscope slide and mixed with 5 µl of 5% eosin B and 5 µl of 10% nigrosin. The stained sperm were spread on the slide, and 400 viable (white) and nonviable (red) spermatozoa were hand counted under light microscope (x400).
Protein Assay
The protein content of the samples was measured by weighing freeze-dried purified proteins on a Cahn microbalance (model C-31; Fisher Scientific, Fairlawn, NJ) or by the modified Lowry procedure [35].
Data Analysis
The data of cholesterol efflux studies were analyzed for significant difference by ANOVA. The significant differences among treatments were determined with the protected Fisher least significant difference test within each incubation time. A value of P < 0.05 was considered statistically significant. The data for sperm functions were analyzed for significant differences by a Student t-test on paired observations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To better understand the mechanism of BSP-mediated sperm cholesterol efflux, we tested the effect of P on sperm cholesterol efflux in the presence of the BSP-A1/-A2 proteins. Because ethanol (final concentration of 0.1%) was used as a vehicle for P, in a first set of experiments, we tested the effect of ethanol on sperm cholesterol efflux (Fig. 1A). As observed previously [28], the BSP-A1/-A2 proteins stimulated significantly the sperm membrane cholesterol efflux. Our results indicated that the presence of a small concentration of ethanol in the incubation medium did not stimulate sperm cholesterol efflux and did not modify the efflux potential of the BSP-A1/-A2 proteins, which remained the same (17.2 ± 1.3%, after 6-h incubation) in the presence or absence of ethanol.
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Then labeled epididymal sperm were incubated in the presence of different concentrations of P (0.01–50 µg/ml) in the presence or absence of the BSP-A1/-A2 proteins (40 µg/ml). Figure 1B shows the results obtained when 1 and 10 µg/ml of P were used. P alone did not stimulate sperm membrane cholesterol efflux and did not affect significantly the cholesterol efflux mediated by the BSP-A1/-A2 proteins. With a smaller concentration of P (0.01 and 0.1 µg/ml, data not shown), the results obtained were similar to those obtained with 1 µg/ml of P. A higher concentration of P (50 µg/ml) was toxic to sperm because all the sperm in this sample were not motile after 6 h. In the other samples, the motility of the sperm was not different from control (data not shown).
Effect of P on Bovine Epididymal Sperm Capacitation and AR in the Presence of the BSP-A1/-A2 Proteins
Because P was recognized to stimulate capacitation and/or AR in several species, during efflux studies, an aliquot of each sample was taken at different times to evaluate the sperm capacitation and the sperm AR. Lyso-PC, which induces the AR only in capacitated sperm, was used as an indirect method to measure sperm capacitation (see Materials and Methods). Therefore, the AR induced by lyso-PC in the present context is meant to reflect capacitation. First, we determined the effect of ethanol on sperm capacitation and AR (Fig. 2). The results indicated that continuous exposure of sperm to the BSP-A1/-A2 proteins stimulated bovine sperm capacitation but did not stimulate sperm AR. After a 6-h incubation, the BSP-A1/-A2 proteins stimulated significantly the capacitation (AR induced by lyso-PC) of labeled epididymal sperm up to 2.1-fold as compared with the control. The presence of ethanol in the medium did not stimulate sperm capacitation or AR and did not modify the capacitation potential of the BSP-A1/-A2 proteins.
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Figure 3 shows the percentage of capacitated (AR induced by lyso-PC) and acrosome-reacted sperm obtained when the sperm were incubated (2–6 h) in the presence of P and/or the BSP-A1/-A2 proteins during efflux studies. After 6 h of incubation, P alone failed to stimulate the sperm capacitation or the sperm AR because the stimulations obtained in the presence of P were not different from that of control with or without lyso-PC. However, when the sperm were incubated simultaneously with the BSP-A1/-A2 proteins and P, the percentage of sperm that undergo the AR increased significantly (not induced by lyso-PC, 15.3%–16.8%, after 6 h of incubation), compared with the percentage of AR obtained in the presence of BSP-A1/-A2 proteins alone (8.8 ± 0.1%, after 6 h of incubation). The percentage of sperm that undergo the AR in the presence of P and the BSP-A1/-A2 proteins was identical (4 h, Fig. 3B) or similar (6 h, not significantly different, Fig. 3C) to the percentage of AR obtained in the same conditions but when lyso-PC was added. These values of AR were also similar to the percentage of AR induced by lyso-PC when sperm were incubated with BSP-A1/-A2 proteins alone (17.8 ± 1.1% after 6 h). There was no difference in AR stimulation with 1 or 10 µg/ml of P. Smaller concentrations of P (0.01 and 0.1 µg/ml) also gave similar results (data not shown). Therefore, P did not induce sperm capacitation of bovine epididymal sperm and did not induce the AR unless the sperm were incubated in capacitation conditions (i.e., in the presence of BSP-A1/-A2 proteins).
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Effect of P on Bovine-Ejaculated Sperm Capacitation and Sperm Viability
A long-time incubation in the presence of the BSP-A1/-A2 proteins is not a physiological condition. Moreover, the incubation of epididymal sperm with the BSP-A1/-A2 proteins only and not the complete seminal plasma could affect the sperm response to P. Therefore, in the next experiments, we used bovine-ejaculated sperm to mimic physiological conditions. To verify the effect of P on ejaculated sperm capacitation, the sperm were washed and then incubated continuously with different concentrations of P for 2–6 h (Fig. 4). Within each capacitation time (2, 4, or 6 h), the P did not stimulate the sperm AR either in the presence or absence of lyso-PC. As control, we used heparin to capacitate the ejaculated sperm. After 4 or 6 h of incubation, heparin stimulated 2.7-fold the sperm capacitation (AR induced by Lyso-PC) but did not stimulate the AR.
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During this experiment, we also tested the effect of P on sperm viability (Table 1). After 6 h of incubation, the percentage of sperm viability obtained in the presence of more than 2 µg/ml of P was significantly different from control (medium alone). With 10 µg/ml of P, the percentage of sperm viability decreased by 15.5% (38.5 ± 2.4%, compared with control, 54.0 ± 0.6%).
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Effect of P on Bovine-Ejaculated Sperm AR
For the result shown in Figure 5, the sperm were incubated in the presence or absence of heparin for 6 h, and then lyso-PC or different concentrations of P were added for another incubation of 15 min. As obtained previously with epididymal sperm incubated continuously with the BSP-A1/-A2 proteins, the lyso-PC and P did not induce the AR unless the ejaculated sperm were incubated in the capacitation medium (in the presence of heparin). The effect of P on sperm AR was concentration dependent with a maximum 2.2-fold increase at 2 µg/ml of P after 4 h (Fig. 5B) or 6 h (Fig. 5C) of incubation with heparin. After 6 h of incubation with heparin, the percentage of AR obtained in the presence of 2 µg/ml of P was similar to the percentage of AR obtained with 100 µg/ml of lyso-PC (21.4 ± 4.1% vs. 23.0 ± 3.7%, control mTALP alone, 9.8 ± 2.6%).
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Time-Course Effect of P in Inducing Bovine-Ejaculated Sperm AR
For these experiments, the sperm were preincubated 6 h with heparin to capacitate the sperm and then incubated with P or lyso-PC. In Figure 6, the time-course effect of P and lyso-PC in increasing the percentage of acrosome-reacted sperm is shown. P (1 and 2 µg/ml) induced an increase within 10 min and reached a maximal value at 15 min (P < 0.05). The time course obtained with 2 µg/ml of P was very similar to that obtained with 100 µg/ml of lyso-PC.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethanol, which has been used to dissolve P, did not affect the cholesterol efflux potential of the BSP-A1/-A2 proteins, sperm capacitation, or AR (Figs. 1A and 2). The present results confirm (Fig. 2B) that the BSP proteins alone are sufficient to stimulate sperm capacitation but are not involved in sperm AR (Fig. 2A). In contrast, long-time incubation of P alone did not stimulate sperm capacitation or AR, whatever the concentration used or the kind of sperm used (epididymal or ejaculated, Figs. 3 and 4). However, P stimulates the sperm AR, at similar levels of lyso-PC, when the sperm were incubated in capacitating conditions (in the presence of BSP-A1/-A2 proteins or heparin, Figs. 3 and 5). The effect of P on AR was rapid and concentration dependent (Fig. 6). The percentage of AR obtained when 1–2 µg/ml of P was used is insignificantly different from that induced by 100 µg/ml of lyso-PC (the concentration generally used to induce the AR of capacitated bovine sperm). Therefore, in vitro, 2 µg/ml of P can be used instead of lyso-PC to stimulate the AR of capacitated sperm. It should be noted that the dose of P that we have used in all experiments is similar to that present in the cumulus oophorus [7].
Our results indicated that long-time incubation with more than 2 µg/ml of P provoked a slight decrease of sperm viability. The low cytotoxic effect of P on sperm viability has also been observed in human [38]. Indeed, a 4-h incubation of human sperm with 10–1000 µmol/L of P (3–300 µg/ml) decreases the sperm viability by 17%–22% (67%–62% versus 84% for the control). A long-time incubation with P is not necessary because our present results indicated that P is not involved in bovine sperm capacitation but involved only in the stimulation of AR, which required only 15 min of incubation of capacitated sperm with P (Figs. 5 and 6).
To our knowledge, no other studies have been done with P and fresh (epididymal or ejaculated) bovine sperm. Cordoba and Beconi [39] reported that bovine frozen-thawed sperm treated with P stimulated the sperm AR in noncapacitated and capacitated (incubated 15 min in the presence of 60 µg/ml of heparin) sperm. Their results indicated that 10 min of incubation with 1 µM of P induced an elevation of sperm intracellular calcium and induced a percentage of true AR (AR value obtained after the treatment minus the AR value obtained at time zero) of 38 ± 5.5% in noncapacitated sperm and of 32 ± 3.5% in capacitated sperm. However, the percentages of AR obtained in the absence of P were not reported. Their results with noncapacitated sperm contradict the present results (Fig. 5). The technique of freezing and thawing, used in their studies, involves several modifications in the sperm plasma membrane (reviewed in [40]). These changes could allow the cryopreserved sperm to respond differently to the steroid.
In vivo, ZP is the physiological inducer of the AR. However, P is present in follicular fluid and the cumulus cells in an appropriate concentration (> 1 µg/ml) [7] to have a biological effect and stimulate the sperm AR. It is hypothesized that the stimulation of some sperm AR by P secreted by cumulus cells releases hydrolytic enzymes that degrade the cells surrounding the oocyte and open the route for the entry of the most potent and viable sperm to acrosome react in contact with the ZP and finally fertilize the ovum [15]. It is also postulated that P primes the sperm to respond to the ZP [9].
In this study, we used long-time incubation of epididymal sperm with BSP proteins to capacitate the sperm. Although the pathway of sperm capacitation (lipid efflux) induced by long-time incubation with the BSP proteins is different from the pathway of capacitation induced by heparin, the response of sperm to P was identical, whatever the method employed to capacitate sperm. P stimulated AR only in capacitated sperm. At this point, we do not know whether the mechanism of AR induction by P is the same for the two methods of capacitation. Because long-time incubation of sperm with the BSP proteins is not a physiological method to capacitate sperm, in the future, it will be more appropriate to study in detail the pathway of AR induction by P when heparin or other physiological capacitating factor (e.g., HDL) was used.
The intracellular signaling cascades induced by P in the stimulation of the AR is not clearly understood. The pathway of P appears to have some similarity with the mechanism of the AR induced by the ZP. One major difference is that P, in contrast to the AR stimulated by ZP, does not involve the activation of a G protein [15, 41]. Most of the available data about the role of P in sperm function have been obtained in human sperm (reviewed in [20, 42]). Briefly, P appears to initiate the human sperm AR via the binding of one or more plasma membrane receptors (different from ZP receptor). P mediates a biphasic increase in intracellular calcium accompanied by depolarization of the membrane potential, but until now it is not clear whether or not voltage-operated calcium channel plays a role in this pathway. Other studies revealed that P (and ZP) activates a calcium-dependent phospholipase C leading to the generation of inositol-trisphosphate and diacylglycerol. P-initiated AR also involves protein phosphorylation.
The use of specific kinase inhibitors in several studies suggested that protein kinase C (PKC), protein tyrosine kinase (PTK) and protein kinase A (PKA) are involved in the P-mediated AR. However, the pathway of P to stimulate the AR has some species specificity. For example, P-mediated AR in human sperm requires the presence of bicarbonate for optimal effect [43] and was abolished by the presence of PKA inhibitors [44]. Whereas in stallion, the action of P in the induction of the AR is additive and independent of the effects of bicarbonate (which also stimulates the AR) [45]. Experiments with protein kinase stimulator and inhibitors suggested that the binding of P appears to lead to the activation of PTK and PKC but did not involve PKA stimulation. In cryopreserved bovine sperm [39], the P stimulation of the AR of heparin-capacitated sperm was inhibited (70%–80%) by the treatments of sperm with methoxyverapamil (voltage-dependent calcium channel inhibitor) and GF-109203X (PKC inhibitor). However, the P induction of the intracellular calcium uptake in heparin-capacitated sperm was only partially (
20%) inhibited by the same treatments. Therefore, in bovine-capacitated cryopreserved sperm, P appears to stimulate the AR through intracellular pathways implicating the PKC and the voltage-dependent calcium channel. In bovine, PKC is localized in the equatorial segment, postacrosomal region of the sperm, and upper region of the acrosome [46]. PKA could also be implicated in the process of AR-mediated by P in bovine because the regulatory subunit isoforms of PKA are found in the head of bovine sperm [47]. Further investigation with fresh ejaculated sperm is necessary to determine the intracellular mechanisms involved in the stimulation of the AR by P.
In conclusion, we have shown that the P does not affect the cholesterol efflux potential of the BSP-A1/-A2 proteins. The results indicated that P is not an inducer of bovine sperm capacitation and stimulates only the AR of capacitated sperm. Therefore, P could be used as a specific inducer of the sperm AR in bovine sperm. Further investigations are required on fresh ejaculated bovine sperm to determine the pathways of the stimulation of the AR by the P.
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FOOTNOTES |
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2 Correspondence: P. Manjunath, Centre de Recherche Guy-Bernier, Hôpital Maisonneuve-Rosemont, 5415 boul. de l'Assomption, Montréal, PQ, Canada, H1T 2M4. FAX: 514 252 3430; puttaswamy.manjunath{at}umontreal.ca
Received: 11 April 2003.
First decision: 11 May 2003.
Accepted: 4 June 2003.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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P < 0.05; 
