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Biology of Reproduction 60, 169-175 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Heparin and High-Density Lipoprotein Mediate Bovine Sperm Capacitation by Different Mechanisms1

M.-E. Lanea, I. Thériena, R. Moreaua, and P. Manjunath2,a

a Departments of Biochemistry and of Medicine, University of Montreal and Guy-Bernier Research Center, Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada H1T 2M4


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Capacitation is an important process in bovine sperm maturation and is an obligatory step prior to fertilization. Two capacitating agents, namely heparin and high-density lipoprotein (HDL), have been shown to induce sperm capacitation. A family of major proteins of bovine seminal plasma designated BSP-A1/A2, BSP-A3, and BSP-30 kDa (collectively called BSP proteins) bind to the sperm surface upon ejaculation via their membrane choline phospholipids. Our previous studies with bovine epididymal sperm showed that BSP proteins potentiate sperm capacitation induced by heparin and HDL. This study was undertaken to clarify the mechanism of capacitation induced by heparin and HDL in the presence of BSP proteins. Washed bovine ejaculated sperm were incubated with heparin (12 µg/ml) or HDL (10–160 µg/ml) in the presence of polyclonal antibodies against purified BSP proteins (anti-BSP proteins). The percentage of capacitated sperm was evaluated after the induction of the acrosome reaction (AR) with lysophosphatidylcholine. When sperm were incubated for 5 h with heparin and anti-BSP proteins (40 µg/ml), the AR level was not significantly different from control levels (16.8 ± 0.9% vs. 12.9 ± 0.9%). In contrast, incubation of sperm for 8 h with HDL and anti-BSP proteins did not inhibit the AR (42.4 ± 1.1% vs. 17.1 ± 1.6 for the control samples). We also investigated the effect of heparin and HDL on protein tyrosine phosphorylation associated with capacitation. The tyrosine phosphorylation of a group of proteins was increased in the presence of heparin. However, HDL did not significantly stimulate protein phosphorylation. The increase in phosphorylation was correlated with an increase in the AR after the incubation with heparin but not with HDL. These results indicate that heparin and HDL mediate capacitation via different mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In mammals, freshly ejaculated sperm are incapable of fertilizing the egg. They gain this ability during their transit through the female reproductive tract, and this process is called capacitation [1–4]. The mechanism of capacitation is poorly understood, but it involves many biochemical changes. These include the removal of adsorbed components from the sperm surface, a change in membrane lipid composition, increased permeability to certain ions such as Ca2+, a change in internal pH, and an increase in plasma membrane fluidity and in metabolism [4–10]. There is also an increased hyperactivation that is believed to result from the redistribution of membrane components during capacitation [4]. Apart from these changes, not much is known. In addition, several studies show that there is a decrease in the membrane cholesterol:phospholipid ratio during capacitation [8, 11]. All of these changes allow the spermatozoa to undergo the acrosome reaction (AR) following interaction with the zona pellucida, the egg's extracellular matrix [12–14].

Many studies have shown that heparin-like glycosaminoglycans (GAG) found in follicular fluid play a role in capacitation of bovine sperm [15–18]. Thus ejaculated sperm incubated with GAG capacitate in a shorter period and then undergo the AR in the presence of the zona pellucida in vivo or in the presence of lysophosphatidylcholine (lyso-PC) in vitro [19]. Lyso-PC induces the AR in capacitated sperm only [19]. It has been postulated that GAG modulate capacitation by binding to proteins of the sperm membrane [20, 21]. In cattle, heparin binds to sperm [22] and induces changes in the intracellular environment of the sperm. This results in Ca2+ uptake and an increase in intracellular free calcium and intracellular pH [9, 10]. Another change associated with heparin-induced capacitation in bovine sperm is an increase in protein phosphorylation [23]. The changes in phosphorylation and cyclic nucleotide levels have also been observed in other sperm functions such as motility and in AR induction [4, 13, 24–26]. Similar observations have been made in sperm of the pig, mouse, and hamster [27–29].

Many studies show that the high-density lipoprotein (HDL) present in follicular and oviductal fluids [30–32] induces sperm capacitation [33]. HDL is the only lipoprotein found in the female genital tract [30–32]. Some studies have shown variations in HDL concentration in the oviduct and follicular fluids during the estrous cycle [11, 30, 34]. Thus, the HDL level is higher during the ovulation period and lower during the remainder of the cycle [11]. The HDL present in the female genital tract facilitate the efflux of cholesterol that occurs during the early steps of capacitation [30, 34]. A recent study shows that the phosphorylation of many sperm proteins is important during heparin-induced bovine sperm capacitation [23]. However, no such studies have been reported using HDL as capacitating agent.

Studies in our laboratory have shown that the seminal plasma proteins are also important agents in sperm capacitation [21, 33]. Bovine seminal plasma (BSP) contains a family of closely related proteins designated BSP-A1/A2, BSP-A3, and BSP-30 kDa (collectively called BSP proteins) [35–39]. These BSP proteins bind to the sperm membrane upon ejaculation, and this binding takes place via choline phospholipids of sperm membrane [40]. They also bind to heparin [41] and HDL [38, 42]. The BSP proteins promote capacitation of bovine epididymal sperm in the presence of heparin [21] and HDL [33].

In this study, we used polyclonal antibodies raised against purified BSP proteins to gain further insight into the mechanism of capacitation induced by heparin and HDL. Our results indicate that these agents mediate sperm capacitation by different mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

BSA (fraction V, fatty acid-free), taurine, L-epinephrine, erythrosin B, flavianic acid (naphthol), heparin (purified from porcine intestinal mucosa), lyso-PC (purified from egg yolk), 3-iso-butyl-1-methylxanthine (IBMX), dibutyryl cAMP (dbcAMP), and Freund's adjuvant were from Sigma Chemical Company (St. Louis, MO); penicillin G and streptomycin sulfate were from Gibco (Burlington, ON, Canada). L-Cysteine, mercuripapain, and iodoacetamide were from ICN (Aurora, OH). Anti-phosphotyrosine antibody (clone 4G10) was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Orthovanadate was from Aldrich Chemical Co. (Milwaukee, WI). ECL (enhanced chemiluminescence) reagent kit was from Mandel Scientific (Boston, MA). Sephadex G-50 and Protein A-Sepharose were from Pharmacia Biotech Inc. (Baie d'Urfé, PQ, Canada). All other reagents used were of analytical grade and obtained from commercial suppliers.

Freshly ejaculated bovine sperm were collected by using an artificial vagina and were obtained from the Centre d'Insémination Artificielle du Québec (St-Hyacinthe, PQ, Canada).

HDL were isolated by density gradient ultracentrifugation from normolipemic human serum as described by Thérien et al. [33]. The purity of HDL was assessed with the Paragon Lipo Gel Kit (Beckman Instruments, Fullerton, CA), and the protein concentration was measured by a modified Lowry technique [43]. Human plasma HDL has been used in human [34] and bovine sperm capacitation studies [33, 44]. HDL isolated from bovine oviductal fluid and plasma has been used in bovine sperm capacitation studies [11, 30]. It should be noted that bovine and human HDL behave similarly and therefore there is no potential problem with interpretation of results [33, 34].

BSP Protein Purification

The BSP was first subjected to alcohol precipitation, and the BSP-A1/A2, -A3, and -30 kDa proteins were isolated by gelatin-agarose affinity chromatography [36]. The adsorbed fractions were further fractionated on a Sephadex G-50 column. The purity of the BSP proteins was confirmed by SDS-PAGE as described further on.

Polyclonal Antibodies

The polyclonal antibodies against BSP proteins were raised in male rabbits as described previously [45, 46]. The antibodies were then purified by affinity chromatography on Protein A-Sepharose. Polyclonal antibodies against platelet activating factor-acetylhydrolase (PAF-AH) were used in control experiments.

Fabs

The antibodies adsorbed on Protein A-Sepharose were treated with mercuripapain for 1 h at 37°C (100:1, w:w) in 150 mM sodium-phosphate buffer, pH 7.0, containing 2 mM EDTA and 10 mM L-cysteine. The reaction was stopped by the addition of iodoacetamide to a final concentration of 25 mM [47]. The Fabs were separated from the Fcs and the intact antibodies by affinity chromatography on Protein A-Sepharose.

Sperm Capacitation and AR

Studies were performed in modified Tyrode's medium as described previously [21, 33, 48]. Ejaculated sperm were washed twice (375 x g, 10 min) with modified Tyrode's albumin lactate pyruvate and were adjusted to a concentration of 1 x 108/ml. Then sperm were incubated at a final concentration of 5 x 107/ml under various conditions: with or without heparin (12 µg/ml) or HDL (10–160 µg/ml) for capacitation; with or without antibodies against BSP-A1/A2, -A3, -30 kDa, or PAF-AH (1.25–160 µg/ml) or Fabs against BSP-A1/A2, -A3, -30 kDa, or PAF-AH (1.25–40 µg/ml) for inhibition studies; and with or without IBMX (100 µM) and dbcAMP (1 mM) for phosphorylation studies. The incubation was carried out in 11 x 75-mm culture tubes for 5 h with heparin or 8 h with HDL at 39°C in a humidified environment of 5% CO2 [49]. At the end of incubation, lyso-PC was added at 100 µg/ml, and the sperm were incubated for an additional 15 min. This concentration of lyso-PC had been previously shown to induce AR in capacitated sperm while having no effect on noncapacitated sperm [19]. 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 [50]. The viability of sperm was determined by staining the cells with eosine-nigrosine [51] and observation under the microscope. In all of our studies on capacitation, sperm viability was between 65% and 70%.

Phosphorylation Studies

After capacitation, 100-µl aliquots of the sperm suspensions were subjected to centrifugation (10 000 x g, 3 min) at room temperature, and the sperm pellet was washed in 1 ml PBS containing 0.2 mM orthovanadate [23]; the sperm pellet was then resuspended in sample buffer without mercaptoethanol and boiled for 5 min [52]. After centrifugation (10 000 x g, 3 min) the supernatant was removed and boiled in the presence of 5% mercaptoethanol in sample buffer for 5 min; it was then subjected to SDS-PAGE as described below.

SDS-PAGE and Immunoblotting

The SDS-PAGE was performed on a 10% gel for the phosphorylation studies and on a 15% gel for the verification of polyclonal antibodies. The proteins were transferred to Immobilon-P (Mandel Scientific) as described by Towbin et al. [53] and submitted to immunodetection [39] using ECL reagent (Mandel Scientific).

Protein Assay

The protein content of the sample was measured by the Bradford method [54] or by weighing of purified proteins on a Cahn microbalance (model C-31; Fisher Scientific, Fairlawn, NJ).

Data Analysis

The data presented here were analyzed for significant differences by a Student's t-test on paired observations or by the Friedman nonparametric test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Specificity of Antibodies

We first established the specificity of the polyclonal antibodies. The alcohol-precipitated BSP proteins (crude BSP proteins, cBSP) and purified BSP proteins were separated by SDS-PAGE, transferred to Immobilon-P membranes, and probed with purified antibodies. Anti-BSP-A1/A2 recognized only BSP-A1/A2 proteins in the cBSP sample and purified BSP-A1/A2 proteins, not the other types of BSP proteins (-A3 and -30 kDa). Similarly, anti-BSP-A3 recognized only BSP-A3 protein, and anti-30 kDa recognized only the BSP-30 kDa protein (Fig. 1, A–C). In these studies we used the antibodies against PAF-AH [45] as a negative control. The PAF-AH is another protein found in BSP in large concentrations (~0.5 mg/ml). The anti-PAF-AH did not recognize any of the BSP proteins (Fig. 1D).



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  		FIG. 1. Immunoblotting of BSP proteins. Proteins were separated by SDS-PAGE on a 15% gel, transferred to Immobilon-P, and probed with purified antibodies directed against BSP proteins or PAF-AH: A) anti-BSP-A1/A2, B) anti-BSP-A3, C) anti-BSP-30 kDa, and D) anti-PAF-AH. Lane 1, cBSP; lane 2, purified BSP-A1/A2; lane 3, purified BSP-A3; lane 4, purified BSP-30 kDa; lane 5, purified PAF-AH.

Effect of Polyclonal Antibodies or Fabs Against BSP-A1/A2, -A3, -30 kDa on Sperm Capacitation with Heparin

Thérien et al. [21, 33] have shown that the BSP proteins potentiate the sperm capacitation induced by heparin and HDL. In the present study, we evaluated the involvement of BSP proteins in the capacitation induced by these two capacitation agents. We determined the levels of AR induced by lyso-PC when ejaculated sperm were incubated with heparin (12 µg/ml) and increasing concentrations of polyclonal antibodies (0–40 µg/ml) raised against the three different purified BSP proteins. In the absence of antibodies and in the presence of heparin, 38.4 ± 1.1% of sperm underwent the AR (Fig. 2A, outside bars), which correlated with previous results [21]. Without the heparin the control level of the AR (medium alone) was approximately 12.9 ± 0.9%. The percentage of AR gradually decreased with increased concentrations of antibodies against BSP-A1/A2,-A3, -30 kDa (Fig. 2A). At a concentration of 40 µg/ml, AR levels were similar to the control level (16.8 ± 0.9%). In contrast, the addition of PAF-AH antibodies (negative control) resulted in no change in the percentage of AR (36.7 ± 2.5%).



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  		FIG. 2. Effect of polyclonal antibodies or Fabs raised against BSP proteins on sperm capacitation with heparin. Ejaculated sperm (1 x 108/ml) were incubated in the presence or absence of heparin alone (12 µg/ml; outside bars) or in the presence of heparin and antibodies or Fabs against BSP proteins (0–40 µg/ml) for 5 h at 39°C. The sperm were then incubated with lyso-PC (100 µg/ml) for 15 min, and AR was assessed as described in Materials and Methods. A) With antibodies, B) with Fabs. Squares, anti-BSP-A1/A2; triangles, anti-BSP-A3; diamonds, anti-BSP-30 kDa; circles, anti-PAF-AH. The values represent the mean ± SEM of three independent experiments with 200 sperm assayed per sample. Nonsignificant differences for anti-BSP-A1/A2, -A3, -30 kDa vs. control (without heparin).

In order to confirm that the inhibition was due to the interaction between the antibodies and the antigen, and not due to interaction between the Fc portion of the antibodies, we prepared Fabs produced with our antibodies and conducted experiments as described above. The Fabs of the anti-BSP proteins (40 µg/ml) also inhibited the AR (19.5 ± 0.4%), whereas in the presence of the same concentrations of Fabs of PAF-AH, the levels of the AR were 38.7 ± 1.8% (Fig. 2B). The utilization of Fabs instead of complete antibodies allowed us to eliminate possible interactions between the antibodies. The antibodies and the Fabs yielded essentially the same results (Fig. 2).

Effect of Polyclonal Antibodies Against BSP-A1/A2, -A3, and -30 kDa on Capacitation Mediated with HDL

Similar experiments were performed using HDL (160 µg/ml) instead of heparin to induce sperm capacitation. We have shown in our previous studies that 160 µg/ml of HDL is required to induce maximum capacitation in epididymal sperm exposed to BSP proteins [33]. In the absence of antibodies, the AR level was 42.4 ± 1.1% with HDL compared to 17.1 ± 1.6% for the control (Fig. 3A, outside bars). In the presence of HDL and increasing concentrations of polyclonal BSP antibodies, the percentage AR did not change (Fig. 3A). For an antibody concentration equivalent (40 µg/ml) to the one that inhibited AR in the presence of heparin, there was no inhibition with HDL. The results obtained with the antibodies against the BSP proteins were the same as those obtained with antibodies against PAF-AH. The differing results that we obtained with HDL may indicate that the concentration of HDL used was too high or that the concentration of antibodies used was too low. Using lower concentrations of HDL (80 and 40 µg/ml), the percentage AR did not change (43.8 ± 2.7% and 43.4 ± 2.2%) (Fig. 3B). We also used 20 and 10 µg/ml of HDL and obtained the same results (data not shown). Similarly, at higher concentrations of antibodies (80 and 160 µg/ml), no change occurred that would indicate that the antibodies had an effect on the sperm AR (Table 1).



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  		FIG. 3. Effect of antibodies raised against BSP proteins on capacitation with HDL; the same protocol as described for Figure 2 using HDL instead of heparin and incubation for 8 h. A) HDL alone (160 µg/ml; outside bars) or with antibodies; squares, anti-BSP-A1/A2; triangles, anti-BSP-A3; diamonds, anti-BSP-30 kDa; circles, anti-PAF-AH. B) With different concentrations of HDL only (40, 80, 160 µg/ml; outside bars) or HDL (40, 80, 160 µg/ml) with anti-BSP-30 kDa (0–40 µg/ml). Circles, 40 µg/ml; triangles, 80 µg/ml; squares, 160 µg/ml. The values represent the mean ± SEM of three independent experiments with 200 sperm assayed per sample. Significant differences for all antibodies (40 µg/ml) vs. control (without HDL): p < 0.05.


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  		TABLE 1. Assessment of the acrosome reaction (%) with HDL (160 µm/ml) in the presence of different concentrations of antibodies against BSP-30-kDa.a

Studies of Phosphorylation during Capacitation with Heparin or HDL

The purpose of the second part of this study was to verify whether or not the phosphorylation observed during capacitation with heparin, as previously reported [23], also occurs during capacitation with HDL. For this study we incubated ejaculated sperm in the presence of HDL (10 and 160 µg/ml) or heparin (12 µg/ml) and with IBMX (100 µM) and dbcAMP (1 mM). As previously reported [23], IBMX (inhibitor of phosphodiesterase) and dbcAMP enhance tyrosine phosphorylation. After the incubation period of 5 or 8 h with heparin and HDL, respectively, an aliquot of the sperm suspension was removed and centrifuged. The sperm pellet was subjected to SDS-PAGE, and immunodetection of phosphotyrosine was performed. In the presence of heparin, there was an increase in the intensity of the phosphorylation bands (40–96 kDa) that was stimulated by the addition of dbcAMP and IBMX (Fig. 4A). Concomitant AR studies were done for incubation with heparin or HDL (Table 2). The results show that the increase in phosphorylation correlated with the increase in AR levels obtained in the presence of heparin. However, there was no increase in phosphorylation with HDL (Fig. 4B). The only observed increase in phosphorylation occurred after the addition of IBMX and dbcAMP to the sample and not because of the presence of HDL. In this case, the level of AR was not correlated with an increase in tyrosine phosphorylation (Table 2). It should be noted that previous studies [23, 55, 56] have shown that the tyrosine phosphorylation of proteins during capacitation occurs very slowly (2–4 h), in contrast to rapid phosphorylation (< 30 min) in other cell-signaling systems.



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  		FIG. 4. The effects of heparin, HDL, and dbcAMP plus IBMX on protein tyrosine phosphorylation. Ejaculated sperm were incubated in the presence of heparin (0 or 12 µg/ml) or HDL (0 or 160 µg/ml) for 5 or 8 h at 39°C. After these incubations, an aliquot was submitted to SDS-PAGE, transferred to an Immobilon-P membrane, and probed for protein tyrosine phosphorylation. A) Incubation with heparin after 5 h and B) incubation with HDL after 8 h.


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  		TABLE 2. Assessment of the acrosome reaction (%) during phosphorylation studies.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Ejaculated sperm acquire fertilizing ability by interacting with capacitation factors present in the female genital tract. Several studies have shown that incubation with heparin or heparin-like GAG promotes capacitation in bovine sperm [15, 50, 57, 58]. Other studies have shown that the cholesterol efflux mediated by HDL may be an important step in sperm capacitation [34, 58, 59]. BSP contains a family of three heparin-binding proteins (BSP proteins) that increase the number of binding sites for heparin on the sperm surface [41] and thereby potentiate heparin-induced bovine sperm capacitation [21]. These BSP proteins also potentiate HDL-induced bovine sperm capacitation [33] and stimulate sperm cholesterol efflux during ejaculation [59]. However, in each instance the mechanisms involved are not well understood.

In the current study, by using specific polyclonal antibodies against BSP proteins, we showed that heparin and HDL induce capacitation by different mechanisms. The bovine ejaculated sperm capacitation induced by heparin is inhibited by the addition of polyclonal antibodies against BSP proteins. These data correlate with our previous studies indicating that the BSP proteins play an important role in heparin-induced capacitation [21]. The inhibition of sperm capacitation by anti-BSP proteins was dose dependent and reached a maximum at a concentration of 40 µg/ml of antibodies. The specificity of the inhibition was further confirmed by using Fabs instead of polyclonal antibodies. The studies with Fabs indicated that the inhibition was due not to the aggregation of sperm but to the interaction of Fabs with the sperm-bound BSP proteins. Furthermore, the polyclonal antibodies against the BSP proteins did not interact with heparin as revealed by a dot-blot experiment (data not shown). In addition, polyclonal antibodies against PAF-AH did not inhibit capacitation. Taken together, these results indicate that the polyclonal antibodies to BSP proteins prevent heparin binding to BSP proteins on the sperm membrane. Therefore, it appears that in order for sperm to undergo capacitation, heparin must interact with sperm membrane-bound BSP proteins. It is interesting to note that the ejaculated sperm are coated with all three BSP proteins [60] and all three BSP proteins promote heparin-induced capacitation [21]. Therefore, one would expect only partial inhibition of capacitation with polyclonal antibodies against one type of BSP protein. However, in the present investigation, all three antibodies independently inhibited heparin-induced capacitation in ejaculated sperm in comparison to control levels. Therefore, it appears that the binding of antibodies to any one type of BSP proteins prevents the interaction of heparin with the other BSP proteins, possibly by steric hinderance.

The second finding of this study was that anti-BSP proteins do not inhibit HDL-induced capacitation. This absence of inhibition was confirmed by using a high concentration of antibodies (160 µg/ml) and a low concentration of HDL (10 µg/ml). These results indicate that potentiation of HDL-induced capacitation by BSP proteins occurs by a different pathway. Previous studies have shown that HDL induces sperm cholesterol efflux during capacitation [30, 34]. Our recent study indicates that BSP proteins and HDL stimulate sperm cholesterol efflux independently [59]. Although BSP proteins induce a significant cholesterol efflux after sperm are ejaculated and mixed with seminal plasma, this efflux is insufficient to complete capacitation. A second cholesterol efflux induced by HDL in the female genital tract may be essential for completion of capacitation. This second sterol efflux appears to be BSP independent, since polyclonal antibodies against BSP proteins did not inhibit HDL-induced capacitation. It is not known how HDL interacts with the sperm membrane. HDL alone stimulates cholesterol efflux from ejaculated or epididymal sperm and capacitates sperm to levels induced in the presence of heparin [33, 59]; therefore it is unlikely that BSP proteins are involved in HDL-induced capacitation. This is supported by the fact that the antibodies against BSP proteins had no effect on HDL-induced capacitation. Therefore it is likely that HDL exerts its effect by directly interacting with the sperm membrane. It should be noted that in contrast to epididymal sperm, ejaculated sperm require low concentrations of HDL (160 µg/ml vs. 40 µg/ml) for induction of capacitation. This could be the case for the following reason. In the present study, the sperm used were ejaculated, and they were exposed to a higher concentration of BSP proteins (20–40 mg/ml) for a longer period (~1 h; time required for transport of semen from Artificial Insemination Center and sperm preparation). In our previous study, epididymal sperm exposed to 40 µg/ml BSP proteins for 20 min were used. Alternatively, there may be other factors in seminal plasma that also contribute to promotion of capacitation induced by HDL.

The third finding of this investigation was that HDL-induced capacitation, in contrast to heparin-induced capacitation, is not associated with an increase in protein tyrosine phosphorylation. The result with heparin confirms the report by Galantino-Homer et al. [23] showing that heparin-induced capacitation is associated with tyrosine phosphorylation. However, it should be noted that while that group reported a significant change in the levels of AR in the presence of IBMX or cAMP, no such changes were observed in our experiments. Cross talk between the protein kinase A pathways and tyrosine phosphorylation has been reported [23]. Parrish et al. [61] showed that IBMX and certain derivatives of cAMP do not induce capacitation in the absence of heparin, and our results are in agreement with these observations. The increase in tyrosine phosphorylation associated with capacitation has been reported in various species: humans [55, 56], cattle [23, 61], and mice [62]. These studies did not reveal sharp differences between species; however, they suggest similar cascade events in sperm capacitation. Our results were in line with these studies when we used heparin as an inductor. To date, a tyrosine phosphorylation study has not been carried out using HDL as an inductor of capacitation. The current study is the first to show no effect of tyrosine phosphorylation in HDL-induced bovine sperm capacitation. Also, it has never been demonstrated that phosphorylation of tyrosine could be activated in other cells. Studies in many species show that BSA could act as a cholesterol acceptor [5, 8, 62]. The efflux of cholesterol has been shown to increase tyrosine phosphorylation [62]. It is possible that BSA, in addition to stimulating cholesterol efflux, may trigger other pathways that involve tyrosine phosphorylation.

In view of the present results, we propose the following mechanisms for heparin and HDL-induced sperm capacitation in the female reproductive tract. Capacitation in bovine sperm begins as soon as sperm are ejaculated and mixed with seminal vesicle secretions. The BSP proteins, the major products of the seminal vesicles, induce initial cholesterol efflux from sperm and prime them to undergo capacitation after further interaction with either heparin-like GAG or HDL in the oviduct. A portion of the BSP proteins also coat the sperm membrane. With heparin-induced capacitation, the sperm-bound BSP proteins act as heparin receptors. Heparin could interact with the sperm membrane via BSP proteins to induce a series of intracellular events such as an increase in pH, Ca2+, and cAMP. The exact cascade is unknown, but this pathway does not include protein G-like factors that are known to be associated with protein kinase A [28]. With HDL-induced capacitation, HDL in the oviduct could mediate a second cholesterol efflux step and result in further significant alterations in the sperm membrane. This later step in capacitation is BSP independent. Furthermore, the HDL-induced capacitation does not involve the cAMP cascade, since there was no significant increase in the tyrosine phosphorylation. Currently, we are investigating the effect of BSP antibodies on tyrosine phosphorylation. It is also important to establish whether or not pH and Ca2+ alterations occur in addition to cholesterol efflux during HDL-induced capacitation.

In summary, the results indicate that the mechanisms of heparin- and HDL-induced bovine sperm capacitation are obviously different. After ejaculation, the BSP proteins interact with the sperm membrane and stimulate sperm cholesterol efflux. This initial cholesterol efflux primes the sperm to undergo capacitation upon interaction with heparin or HDL. BSP proteins bind to the sperm membrane and increase the heparin-binding sites on the sperm membrane. BSP proteins therefore play an important role both in heparin-induced capacitation and in tyrosine phosphorylation that occurs during heparin-induced capacitation. Any interruption of this interaction between sperm-bound BSP proteins and heparin results in disruption of capacitation. In an alternative pathway, after the initial cholesterol efflux by BSP proteins, HDL mediates a second efflux of cholesterol (BSP protein independent) leading to capacitation but without inducing phosphorylation.


   ACKNOWLEDGMENTS
 
We are grateful to Dr. Kenneth D. Roberts for proofreading the manuscript.


   FOOTNOTES
 
1 This work was supported by a grant from the Medical Research Council of Canada. Back

2 Correspondence: P. Manjunath, Centre de recherche Guy-Bernier, Hopital Maisonneuve-Rosemont, 5415 boul. l'Assomption, Montreal, PQ, Canada H1T 2M4. FAX: 514 252 3430; manjunap{at}ere.umontreal.ca Back

Accepted: September 1, 1998.

Received: July 8, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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