Angiotensin II induces acrosomal exocytosis in bovine spermatozoa

Yael Gur1, Haim Breitbart2, Yehudit Lax2, Sara Rubinstein2, and Nadav Zamir2,3

1 Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv 69978; 2 Department of Life Sciences, Bar-Ilan University, Ramat Gan 52900; and 3 D-Pharm, Kiryat Weizmann, Rehovot 76123, Israel

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Procedures
Results
Discussion
References

Ejaculated mammalian spermatozoa must reside in the female genital tract for some time before gaining the ability to fertilize the egg. During this time, spermatozoa undergo some physiological changes that collectively are called capacitation. Capacitation of mammalian spermatozoa is a prerequisite for acrosome reaction, which is an exocytotic event occurring before fertilization. The specific biophysical and biochemical changes that accompany sperm capacitation and the agonists inducing acrosome reaction are not fully understood. Using SDS-gel electrophoresis and immunoblotting, we demonstrate the existence of a class of angiotensin receptors (AT1) in bovine spermatozoa. In capacitated sperm, we show that angiotensin II (ANG II) AT1 receptors are localized in the head and tail, whereas in noncapacitated cells the receptors are localized in the tail only. We find that ANG II markedly stimulates acrosomal exocytosis of capacitated bovine spermatozoa in vitro in a concentration range of 0.1-10 nM. No effect of ANG II was found in noncapacitated cells. The ability of ANG II to stimulate the acrosome reaction depends on the presence of calcium ions in the incubation medium. The ANG II-induced acrosome reaction was markedly inhibited by a selective AT1 receptor antagonist, losartan (DUP 753). PD-123319, a selective antagonist of the ANG II AT2 receptor, had no effect on the ANG II-induced acrosome reaction. Thus ANG II via activation of AT1 receptors may play a regulatory role in the induction of the acrosome reaction.

angiotensin II; capacitation; acrosomal exocytosis; AT1 receptors

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Procedures
Results
Discussion
References

ONCE THE SPERMATOZOON is tightly bound to the zona pellucida surface of the egg, there is an initiation of a signal transduction cascade that precipitates the acrosome reaction (15). The acrosome reaction involves fusion and fenestration of the outer acrosomal membrane with the overlying sperm plasma membrane, resulting in the release of the acrosomal hydrolytic enzymes (5, 31). The acrosome reaction enhances the spermatozoon penetration through the egg's zona pellucida and its subsequent fusion with the egg's vitelline membrane (31). The acrosome reaction is essential for successful fertilization (15, 31). Elucidation of the mechanisms regulating acrosome reaction is therefore important for understanding mammalian fertilization. A variety of agonists derived from the egg's extracellular coat or zona pellucida or constituents of the female reproductive tract affect sperm function (30, 35) and may trigger the acrosome reaction of mammalian spermatozoa via receptor-mediated mechanisms (30). The zona pellucida-derived glycoproteins can initiate the acrosome reaction in vitro and are generally considered to be the in vivo inducers of the acrosome reaction (2, 30). However, the mammalian acrosome reaction can also be initiated in vitro by other agonists, such as progesterone (3, 26), prostaglandins (13), atrial natriuretic peptide (27, 36), and epidermal growth factor (18). These agonists may have a direct and/or synergistic effect on the zona pellucida (26). ANG II may be a candidate for induction of the acrosome reaction.

ANG II is a hormone that exerts a wide range of physiologically important effects on various tissues by interacting with cell surface receptors (9, 28). Two major subtypes of receptors (AT1 and AT2) have been distinguished and characterized by pharmacological and molecular biology techniques. AT1 receptors mediate most of the known functions of ANG II (9, 28). AT2 receptors may function during development (9, 28). Pharmacologically, the AT1 receptors have selective affinity for biphenylimidazoles, such as losartan, and insensitivity to tetrahydroimidazopyridines, typified by PD-123319 and ANG III (9, 28). The second class of ANG II receptors, the AT2 receptor, has a high affinity for tetrahydroimidazopyridines, such as PD-123319, and a very low affinity for biphenylimidiazoles, such as losartan (9, 28).

Several lines of evidence suggest that endogenous ANG II may act on the mammalian spermatozoa. ANG II is synthesized in the rat and human ovaries (12, 23) and also exists in follicular fluids (7, 11). In addition, high-affinity ANG II AT1 receptors have been localized in the tails of ejaculated human and rat spermatozoa (29). ANG II, at low concentration, enhanced motility of mammalian spermatozoa in vitro (29). Because ANG II has been implicated in sperm function, we further investigated the involvement of ANG II in the induction of the acrosome reaction in capacitated bovine spermatozoa. In addition, we examined the involvement of the ANG II receptors in the ANG II-induced acrosome reaction.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Procedures
Results
Discussion
References

Human ANG II, ANG III, and ANG IV were purchased from Peninsula Laboratories (Belmont, CA). The calcium ionophore A-23187 free acid was from Calbiochem.

The AT1 antagonist losartan (DUP 753; 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)-methyl]imidazole, potassium salt) was obtained from Du Pont-Merck (Wilmington, DE).

The AT2 competitor PD-123319 (5-(1-[4-(dimethylamino)-3-methylphenyl) methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c] pyridine-6-carboxylic acid,difluoroacetate monohydrate) was purchased from Parke, Davis (Ann Arbor, MI).

AT1 (N-10) is an affinity-purified rabbit polyclonal antibody raised against amino acids 15-24, mapping to the amino terminus of the ANG II AT1 receptor of human origin (identical to the corresponding rat sequence) (22). AT1 (N-10) reacts with the ANG II AT1 receptor of mouse, rat, and human origin by Western blotting, immunoprecipitation, and immunohistochemistry. It does not cross-react with the ANG II AT2 receptor. AT1 (N-10) (SC-1173) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Control (antigenic) peptide for competition studies (SC-1173-P) was also purchased from Santa Cruz Biotechnology.

Bovine sperm cells were obtained from the Artificial Insemination Service, Hafez Haim, Israel. All the other reagents were purchased from Sigma Chemical (St. Louis, MO).

    EXPERIMENTAL PROCEDURES
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Materials & Methods
Procedures
Results
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References

Sperm preparation. Bovine sperm cells were collected in an artificial vagina and diluted (1:1, vol/vol) in NKM medium, pH 7.4, containing 110 mM NaCl, 5 mM KCl, and 10 mM MOPS. The cells were washed and centrifuged three times at 780 g for 10 min in NKM medium, and the final pellet was resuspended in NKM with the sperm concentration adjusted to 1-3 × 109 cells/ml.

Capacitation and acrosome reaction evaluation. In vitro capacitation was accomplished by the method of Parrish et al. (24). Briefly, washed sperm cells (108 cells/ml) were capacitated for 4 h at 37°C in glucose-free modified Tyrode solution (mTALP medium), containing 100 mM NaCl, 3.1 mM KCl, 25 mM NaHCO3, 0.29 mM KH2PO4, 21.6 mM Na lactate, 1.5 mM MgCl2, 0.1 mM sodium pyruvate, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 10 IU/ml penicillin (pH 7.4), 50 µg/ml BSA (fraction V), and 20 µg/ml heparin. The capacitated sperm (108 cells/ml) were incubated for an additional 20 min with CaCl2 (2 mM) in the presence of various agents and/or hormones.

At the end of the incubation period, the cells were pelleted by centrifugation (12,930 g for 5 min), and the occurrence of the acrosome reaction was determined by measuring the activity of acrosin released in the supernatant fluid, as previously described for bovine spermatozoa (18). Briefly, the supernatant fluid was adjusted to pH 3.0 with 3 M HCl, and the acrosin activity was measured by the esterolytic assay with benzoylarginine ethyl ester (BAEE) as substrate by recording the increase in absorbance at 259 nm with time (acrosin activity: nanomoles of BAEE hydrolyzed by 108 cells per minute). The molar absorption coefficient is 1,150. All the values are given after the spontaneous acrosin release was subtracted. The occurrence of the acrosome reaction was confirmed morphologically by staining sperm cells with horseradish peroxidase (HRP)-conjugated Pisum sativum agglutinin (PSA) (20).

Whole cell lysates. Proteins from sperm cells were extracted as described (19). Briefly, washed sperm cells (108 cells) were solubilized in SDS-lysis buffer containing 125 mM Tris (pH 7.5), 4% SDS, 1 mM sodium orthovanadate, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride added just before use. Cells were lysed for 10 min at room temperature and centrifuged at 12,930 g for 5 min at 4°C. The supernatant fluid was supplemented with 0.05% bromophenol blue, 5% glycerol, and 2% beta -mercaptoethanol and boiled for 5 min.

Immunoblot analysis. For immunoblotting, proteins of equivalent cell amounts (108 cells) were separated on 10% SDS-polyacrylamide gels and then electrophoretically transferred to nitrocellulose membranes (200 mAmp; 1 h) by use of a buffer composed of 25 mM Tris (pH 8.2), 192 mM glycine, and 20% methanol.

For Western blotting, nitrocellulose membranes were blocked with 5% BSA in Tris-buffered saline (TBS), pH 7.6, containing 0.1% Tween 20 (TBST), for 30 min at room temperature. The membranes were incubated overnight at 4°C in the presence of an antibody against the AT1 receptor (N-10), diluted 1:1,000.

Next, the membranes were washed three times with TBST and incubated for 1 h at room temperature with specific HRP-linked secondary antibody (Jackson Laboratories, West Grove, PA) diluted 1:15,000 in TBST. The membranes were washed three times with TBST and visualized by enhanced chemiluminescence (Amersham, Little Chalfont, UK). Specificity of the AT1 receptor antibody was determined by preabsorbing the antibody with 10 µg of its peptide antigen for 1 h before incubating the antibody with the membrane.

Immunocytochemistry. Sperm cells (106 cells) were spread on glass coverslips and then fixed and permeabilized with cold methanol (30 s). Nonspecific reactive sites were blocked with 1% BSA in TBS for 10 min at room temperature. The cells were incubated for 60 min at 37°C with the AT1 (N-10) antibody diluted 1:1. The second antibody, FITC-conjugated rabbit polyclonal antibody, was diluted 1:100 in 1% BSA TBS for 10 min in a dark box. Between antibody incubations, cells were washed three times (5 min) in TBS.

The cells were then examined with an Olympus photomicroscope (Vanox AHBT3; Olympus, Lake Success, NY) and photographed with Kodak 200 ASA film (Eastman Kodak, Rochester, NY) and a ×40 objective. Nonspecific staining was determined by incubation in the presence of the antigenic peptide (10 µg).

Data analysis. Results are shown as means ± SE. Statistical analyses were performed using the paired Student's t-test or one-way analysis of variance, followed by multiple comparisons using the least significant difference method (see Fig. 3). Statistical significance was defined as P < 0.05.

    RESULTS
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Materials & Methods
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Results
Discussion
References

Expression of ANG II AT1 receptors in capacitated bovine spermatozoa. To confirm the presence of AT1 receptors, SDS-extracted cells were subjected to Western immunoblot analysis with the AT1 receptor-specific antibody. The AT1 receptor antibody identified a single protein band with an approximate molecular mass of 41 kDa (Fig. 1), which is typical of AT1 receptors from many mammalian cell types (9). The specificity of the antibody was demonstrated by preabsorption with the peptide antigen, which completely prevented immunodetection of the 41-kDa protein band (Fig. 1).


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Fig. 1.   Expression of bovine sperm angiotensin receptor (AT1) in capacitated cells. Protein immunoblots showing AT1 receptor in bovine sperm. Total protein was extracted with SDS-PAGE, transferred to nitrocellulose, and immunoblotted with AT1 receptor-specific polyclonal antibody in the absence (-) or presence (+) of specific antigenic peptide (pep), as described in MATERIALS AND METHODS. Molecular weights of prestained high-range marker proteins are indicated (×10-3). Blot shown is representative of 3 separate experiments with sperm from different bulls.

Immunocytochemical localization of ANG II AT1 receptors in sperm cells. The AT1 receptor-specific antibody (N-10) was used to visualize AT1 receptors by immunocytochemistry in capacitated and noncapacitated bovine spermatozoa. Immunostaining of AT1 receptors was seen in the tail of noncapacitating bovine sperm cells (Fig. 2A). Interestingly, a different distribution of AT1 receptors was observed in capacitated sperm cells, which show intense staining of the postacrosomal region and along the tail (Fig. 2B). Preabsorption of the AT1 receptor-specific antibody with the immunizing peptide abolished the immunostaining in both head and tail of capacitated cells and in the tail of noncapacitated cells (data not shown).


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Fig. 2.   Immunocytochemical localization of the AT1 receptor in bovine sperm cells. Capacitated and noncapacitated bovine sperm cells were fixed and stained with AT1 receptor-specific antibody, as described in MATERIALS AND METHODS. Staining was observed in tail of noncapacitated sperm cells (×400) (A) and in tail and postacrosomal region of capacitated cells (×400) (B). Similar results were seen with sperm from 6 different bulls in each experiment.

Induction of acrosomal exocytosis by ANG II. The acrosome reaction was determined by measuring the release of acrosin from bovine spermatozoa and by staining with PSA. Addition of ANG II in doses ranging between 0.1 and 10 nM for 20 min to capacitated bovine spermatozoa resulted in significant enhancement of acrosin release compared with untreated cells (Fig. 3). Maximal stimulation of acrosin release (fivefold increase compared with untreated cells) was observed at 1 nM of ANG II (Fig. 3). However, at higher concentrations of ANG II, the stimulatory effect was less pronounced. Addition of ANG II at doses of 1, 10, and 100 nM for 20 min to noncapacitated bovine spermatozoa had no stimulatory effect on acrosin release compared with untreated noncapacitated cells (data not shown).


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Fig. 3.   Dose-response curve for effect of angiotensin (ANG) II on acrosin release from capacitated bovine spermatozoa. Bovine sperm (108 cells/ml) were capacitated for 4 h in mTALP medium containing heparin, followed by 20 min of incubation in the presence of 2 mM CaCl2 and increased concentrations of ANG II. Activity of released acrosin from cells was determined (see MATERIALS AND METHODS for details) and was given as nmol benzoylarginine ethyl ester (BAEE) · 108 cells-1 · min-1. Acrosin activity in the absence of Ca2+ was subtracted from each point. Values are means ± SE of duplicate determinations from >= 10 different bulls. Different letters above bars, significant difference (P < 0.001).

Comparative evaluation of the acrosome reaction induced by ANG II and A-23187 as assessed by acrosin release and sperm staining. The acrosin release served as an index for acrosomal exocytosis in capacitated bovine spermatozoa. ANG II (1 nM) and the calcium ionophore A-23187 (10 µM) potently stimulated acrosin release (Fig. 4). The acrosin release assay was correlated with acrosome-reacted cells as evaluated by staining spermatozoa with HRP-conjugated PSA (20).


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Fig. 4.   Comparative evaluation of acrosome reaction induced by ANG II (1 nM) and A-23187 (10 µM) as assessed by acrosin release (solid bars) or sperm staining (open bars). Sperm capacitation and induction of acrosome reaction were performed as described in Fig. 3. Sperm staining was performed with horseradish peroxidase (HRP)-conjugated Pisum sativum agglutinin (PSA) (20). Value of 1 (control) is defined as number of capacitated bovine spermatozoa that underwent acrosome reaction in the presence of CaCl2 (2 mM) in medium and in the absence of inducer. This value applies to 6% of cells undergoing acrosomal exocytosis, as evaluated by sperm staining. Activity of acrosin released from these cells was 32 nmol BAEE · 108 cells-1 · min-1. Data are from 4 different experiments with sperm from 4 bulls.

The range of the effect observed with the Ca2+ ionophore is in good agreement with the induction of acrosomal exocytosis of capacitated bovine spermatozoa by the zona pellucida (8). The magnitude of acrosome reaction induced by ANG II reached ~50% of that induced by A-23187 (10 µM, Fig. 4).

Role of calcium in ANG II-induced acrosomal exocytosis. Typically, acrosomal exocytosis is a calcium-dependent process (15, 30, 31). We examined the role of extracellular calcium ions by exposure of capacitated bovine spermatozoa to ANG II in Ca2+-free medium in the presence of LaCl3, which is known to block Ca2+ influx effectively. We found that the stimulatory effect of ANG II on acrosomal exocytosis was completely abolished when cells were incubated in medium without added Ca2+ and in the presence of LaCl3 (1 mM) (Fig. 5). These data imply that ANG II-induced acrosomal exocytosis requires extracellular Ca2+.


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Fig. 5.   Calcium dependence of ANG II-induced acrosin release from capacitated bovine spermatozoa. Effect of ANG II (1 nM, solid bars) on acrosin release was tested in the presence of CaCl2 (2 mM) or Ca2+-free medium containing LaCl3 (1 mM). Acrosin activity is expressed as described in Fig. 3. Values are means ± SE of duplicates from 7 different bulls. Results are significantly different (*P < 0.05) from appropriate control not treated with ANG II (open bars).

Effects of ANG II and ANG II fragments (ANG III and ANG IV) on acrosomal exocytosis. We tested the ability of ANG II and of ANG II fragments (ANG III and ANG IV) to induce acrosomal exocytosis of capacitated bovine spermatozoa. The primary structures of ANG II and of ANG II fragments are shown in Table 1. Whereas ANG II is a potent inducer of acrosomal exocytosis, as shown before (Fig. 3), ANG III and ANG IV failed to induce acrosomal exocytosis at doses of 1, 10, and 100 nM (Fig. 6). These results indicate that NH2-terminal amino acid residues of ANG II are essential for its biological effect on sperm cells.

                              
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Table 1.   Primary structures of ANG II, ANG III and ANG IV


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Fig. 6.   Dose-response curves for effects of ANG II (black-square), ANG III (bullet ), and ANG IV (black-triangle) on acrosin release from capacitated bovine spermatozoa. Sperm capacitation and induction of acrosome reaction were performed as described in Fig. 3. Acrosin activity is also as described in Fig. 3. Values are means ± SE; n = 3 for each ANG II fragment.

ANG II-induced acrosome reaction is receptor mediated. We studied the effects of selective AT1 and AT2 receptor antagonists on acrosomal exocytosis induced by ANG II in capacitated bovine spermatozoa. Losartan (50 nM), a selective AT1 receptor antagonist, markedly (70%) inhibited the ANG II-induced acrosome reaction in our model system (Fig. 7). Higher concentrations of losartan (100 and 1,000 nM) had similar inhibitory effects (data not shown). Losartan (50 nM) by itself had no effect on the acrosome reaction in the absence of ANG II (Fig. 7). On the other hand, PD-123319, a selective AT2 receptor antagonist at concentrations of 1, 5 and 10 nM, had no effect on the ANG II-induced acrosome reaction in capacitated bovine spermatozoa (Table 2). These results suggest that ANG II-induced acrosomal exocytosis is mediated by activation of the AT1 receptors in capacitated sperm cells.


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Fig. 7.   Effect of ANG II AT1 receptor antagonist losartan on ANG II-induced acrosome reaction of capacitated bovine spermatozoa. ANG II (1 nM) and losartan (Los, 50 nM) were added alone or in combination to capacitated bovine spermatozoa. Activity of released acrosin from cells was determined as described in Fig. 3. * Significantly (P < 0.05) different from ANG II (n = 10).

                              
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Table 2.   Effect of ANG II AT2 receptor antagonist PD-123319 on ANG II-induced acrosin release from capacitated bovine spermatozoa

    DISCUSSION
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Abstract
Introduction
Materials & Methods
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Results
Discussion
References

In mammals, the acrosome reaction is regulated by agonists originating from the egg or its associated cellular and acellular structures, or from the female reproductive tract (15, 30, 31).

The present study suggests for the first time that ANG II could serve as such an agonist. Previous studies showed that ANG II is synthesized in mammalian ovaries (12, 23), and ANG II receptors have been localized in rat and bovine ovarian follicles, specifically the granulosa cell layers and the theca interna (12). The presence of ANG II and its receptors within the ovary supports a role for ANG II in ovarian function. Many studies have demonstrated that ovarian ANG II may play an important role in regulation of ovarian steroidogenesis (14, 25), oocyte maturation (32, 33), ovulation (1, 16, 32, 34), and corpus luteum formation (21). Moreover, ANG II is secreted into follicular fluid, and its concentration is >10 times greater than that found in the plasma (7, 11). ANG II derived from the ovary may therefore act on spermatozoa and affect their function.

Recently, Vinson et al. (29) by an immunocytochemical method visualized AT1 receptors in the tails of ejaculated rat and human spermatozoa. These AT1 receptors are functional, because exposure of human spermatozoa for 5 min to a low concentration of ANG II amide increased both the percentage of human motile sperm and their linear velocity, whereas a selective AT1 receptor antagonist, losartan, markedly inhibited the action of the ANG II amide on the percentage of motile human spermatozoa (29).

The present results obtained from experiments carried out with capacitated bovine spermatozoa in vitro further support and extend a functional role for ANG II before and during fertilization. Using a specific antibody against the AT1 receptor, we visualized the AT1 receptor in the postacrosomal region and tail of capacitated bovine spermatozoa. We verified the existence of the AT1 receptor by immunoblotting. Structurally, the AT1 receptor belongs to the seven transmembrane domain family of G protein-coupled receptors. The receptor is a 359-amino acid long protein of a molecular mass of ~41 kDa. Our results demonstrate the existence of the native AT1 receptor in capacitated bovine spermatozoa. The native AT1 receptor, however, is probably subject to posttranslational modifications, because its extracellular sequences contain multiple N-glycosylated sites. This is consistent with the observation that the molecular mass of the protein determined by gel electrophoresis is dependent on the degree of glycosylation (9). Indeed, Vinson et al. (29) reported the existence of a 60-kDa glycosylated AT1 receptor in ejaculated human spermatozoa.

We found that ANG II induced the acrosome reaction of capacitated bovine spermatozoa but had no effect on the acrosome reaction of noncapacitated bovine spermatozoa. As observed previously in ejaculated (noncapacitated) sperm in rats and humans (29), we also found AT1 receptors localized to the tail region in noncapacitated bovine sperm cells. This result is consistent with the increased sperm motility induced by ANG II in noncapacitated cells reported by Vinson et al. (29). In contrast, in capacitated bovine sperm cells we observed AT1 receptors in the postacrosomal region of the head in addition to those present in the tail. Our results clearly demonstrate that activation of these receptors in the head are involved in the acrosome reaction. The mechanism underlying these changes in the distribution of these receptors during capacitation is not clear.

To examine whether the ANG II-induced acrosome reaction is a calcium-dependent process, we tested its effects on capacitated bovine spermatozoa in the presence of the nonselective calcium channel blocker LaCl3 in calcium-free medium. Under these experimental conditions, ANG II was ineffective, indicating the importance of extracellular Ca2+ to ANG II action.

Induction of acrosomal exocytosis of capacitated bovine spermatozoa by a very low concentration of ANG II (0.1 nM) is in good agreement with Michaelis-Menten kinetic Kd values (~0.1 nM) obtained for ANG II binding to different somatic cell types (4) and also with the physiological concentrations of ANG II observed in follicular fluid (7, 11). These observations support a putative physiological role for the hormone in the regulation of acrosomal exocytosis.

The effect of ANG II on bovine spermatozoa is specific and mediated by plasma membrane AT1 receptors. This is evidenced by the inhibition of the stimulatory effect of ANG II on the acrosome reaction by the selective AT1 receptor antagonist losartan, whereas the AT2 selective antagonist PD-123319 had no inhibitory effect on this stimulation. The fact that losartan inhibited ~70% of ANG II-induced acrosin release in our experimental model does not exclude partial involvement of atypical AT receptors apart from AT1 receptors. Indeed, Chaki and Inagami (6) described a unique ANG II receptor subtype in mouse neuroblastoma Neuro-2A cells that has a high affinity for ANG II, a negligible affinity for ANG III, and an insensitivity to losartan and PD-123319 at micromolar concentrations. To date, these ANG II receptors, termed Neuro-2A AT receptors, have been described only in cell lines, and their presence in sperm cells should be examined.

Acrosomal exocytosis of mammalian sperm induced by agonists such as ANG II is analogous to exocytosis seen in somatic endocrine cells or neurons. Indeed, ANG II was shown to be a potent inducer of secretion from somatic endocrine cells and neurons. ANG II binds to AT1 receptors localized in sympathetic nerve terminals and enhances the release of norepinephrine (4). It also activates AT1 receptors of glomerulosa cells of the adrenal cortex and stimulates the release of aldosterone (4). Thus the functions of ANG II in somatic cells and in sperm cells share a common feature, namely, induction of exocytosis.

In conclusion, our present study has shown that ANG II may regulate the acrosome reaction via activation of AT1 receptors. These results, together with the previous observations that ANG II enhances sperm cell motility, suggest that ANG II may play an important role in mechanisms preceding fertilization in mammals.

    ACKNOWLEDGEMENTS

This study was presented in preliminary form at the Israeli Society of Physiology and Pharmacology in Maale Hahamishah, Israel, in October 1996 (10).

    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: H. Breitbart, Dept. of Life Sciences, Bar-Ilan Univ., Ramat-Gan 52900, Israel.

Received 12 January 1998; accepted in final form 8 April 1998.

    REFERENCES
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Abstract
Introduction
Materials & Methods
Procedures
Results
Discussion
References

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