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
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ABSTRACT |
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 |
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 |
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 |
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%
-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 |
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.
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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.
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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).
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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.
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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).
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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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Fig. 6.
Dose-response curves for effects of ANG II ( ), ANG III ( ), and
ANG IV ( ) 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.
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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
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 |
DISCUSSION |
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.
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