Atrial natriuretic peptide induces acrosomal exocytosis of
human spermatozoa
Ronit
Rotem,
Nadav
Zamir,
Nurit
Keynan,
Dalit
Barkan,
Haim
Breitbart, and
Zvi
Naor
Department of Biochemistry, George S. Wise Faculty of Life
Sciences, Tel Aviv University, Ramat Aviv 69978; and Department of
Life Sciences, Bar-Ilan University, Ramat Gan 52900, Israel
 |
ABSTRACT |
Acrosomal exocytosis
in mammalian spermatozoa is a process essential for fertilization. We
report here that atrial natriuretic peptide (ANP) markedly stimulates
acrosomal exocytosis of capacitated human spermatozoa. Typically, ANP
exerts some of its actions via activation of the ANP receptor (ANPR-A),
a particulate guanylyl cyclase-linked receptor, and subsequent
formation of guanosine 3',5'-cyclic monophosphate (cGMP).
We found that ANP-stimulated acrosome reaction was inhibited by the
competitive ANPR-A antagonist anantin, indicating a receptor-mediated
process. A linear fragment of ANP, ANP-(13
28), and another ANP-like
compound, brain natriuretic peptide, were inactive. The stimulatory
effect of ANP on acrosome reaction was mimicked by the permeable cGMP
analog, 8-bromo-cGMP (8-BrcGMP). Addition of the protein kinase C (PKC)
inhibitors, staurosporine and GF-109203X, resulted in a dose-related
inhibition of ANP-induced acrosome reaction. Also, downregulation of
endogeneous PKC activity resulted in inhibition of ANP- but not
8-BrcGMP-induced acrosome reaction. Removal of extracellular
Ca2+ abolished ANP-induced
acrosome reaction. Thus ANP via
Ca2+ influx, PKC activation, and
stimulation of particulate guanylyl cyclase may play a role in the
induction of acrosome reaction of human spermatozoa.
acrosome reaction; guanosine 3',5'-cyclic
monophosphate; calcium; protein kinase C
 |
INTRODUCTION |
AT THE TIME OF FERTILIZATION, capacitated mammalian
spermatozoa undergo an exocytotic process termed acrosome reaction (28, 30). The acrosome reaction involves fusion between the outer acrosomal
membrane and the overlying plasma membrane, leading to the exposure and
release of acrosomal hydrolytic enzymes (28, 30). The acrosome reaction
enables the sperm cell to penetrate the zona pellucida and to fuse with
the egg's plasma membrane (28, 30). The acrosome reaction is therefore
a prerequisite for successful fertilization. Thus elucidation of the
mechanism regulating acrosome reaction is important for understanding
human fertilization.
Several lines of evidence suggest that agonists derived from the egg's
extracellular coat or zona pellucida or constituents of the female
reproductive tract may trigger the acrosome reaction of capacitated
spermatozoa in a receptor-mediated mechanism (30). Atrial natriuretic
peptide (ANP) may be a candidate for induction of the acrosome
reaction. ANP was detected in rat ovaries (8, 13, 14), oocytes (15),
and follicular fluids (2, 24, 25). In addition, high-affinity binding
sites for ANP were localized in human spermatozoa (23). We have
previously demonstrated that ANP-induced chemokinesis and chemotaxis of
capacitated human spermatozoa (32). More recently, it was reported that
ANP induces an acrosome reaction of human (2, 5) and bovine spermatozoa
(31), but only guanosine 3',5'-cyclic monophosphate (cGMP)
was implicated as a mediator in ANP action (2, 31). The present study
was undertaken to further elucidate the mechanism underlying
ANP-induced acrosome reaction of capacitated human spermatozoa in
vitro. We have recently demonstrated the presence of protein kinase C
(PKC) in mammalian sperm and its possible involvement in sperm motility and acrosome reaction (6, 11, 16, 20-22). We therefore examined here the possible involvement of PKC in ANP-induced acrosome reaction. We propose that ANP-induced acrosome reaction is mediated by
Ca2+, PKC, and cGMP.
 |
MATERIALS AND METHODS |
ANP-(1
28) (human), ANP-(5
28) (human, rat), ANP-(13
28) (rat), and
porcine brain natriuretic peptide (BNP) were purchased from Peninsula
Laboratories (Belmont, CA). A-23187, 8-bromo-cGMP (8-BrcGMP),
12-O-tetradecanoylphorbol
13-acetate (TPA), and rose bengal were purchased from Sigma Chemical
(St. Louis, MO). Bismark brown was from Searle Diagnostics.
Staurosporine was purchased from Kyowa Medox (Tokyo, Japan). Trypan
blue was purchased from Fluka (Switzerland) and anantin from Bachem
(Bubendorf, Switzerland). GF-109203X was purchased from Calbiochem (La
Jolla, CA).
Preparation of spermatozoa.
Human sperm were obtained from fresh ejaculates of healthy donors
(22-30 yr old) after 72 h of abstinence. Samples were allowed to
liquefy at room temperature for 30-60 min. Sperm cells were then
washed twice (750 g for 10 min) with
Ham's F-10 medium containing 0.5% of human serum albumin (HSA) and
incubated for 2.5 h at a temperature of 35°C for capacitation.
Acrosomal reaction evaluation.
Capacitated sperm cells (5 × 108 cells/aliquot) were incubated
for 60 min with the various hormones and/or drugs at 35°C.
Acrosomal sperm status was analyzed by the triple-stain technique, as
described by Talbot and Chacon (26). Sperm were resuspended in Ham's
F-10 medium without HSA, containing trypan blue (1% in
phosphate-buffered saline), for 15 min. Samples were centrifuged (750 g for 5 min) and washed with saline
until the stain disappeared. Sperm were then fixed in glutaraldehyde
(3%, in cacodylate buffer) for 30 min at room temperature and later
washed twice with distilled deionized
H2O
(ddH2O), resuspended in 50 µl of
ddH2O, and pipetted onto a
microscope glass slide. The air-dried slides were incubated in bismark
brown (0.8%) for 8 min at 40°C. Slides were again washed in
ddH2O to remove excess stain and
incubated in rose bengal (0.8%) for 25 min at room temperature. Slides
were washed to remove excess stain, passed twice through absolute
ethanol alcohol dehydration, and cleared twice in xylene (100%). Sperm
were then examined by light microscopy under oil immersion to follow
the acrosome status (22).
Data analysis.
Results are shown as means ± SE. Statistical analyses were
performed using paired Student's
t-test. Statistical significance was
defined as P < 0.05.
 |
RESULTS |
Induction of acrosome reaction by ANP.
The acrosome reaction was detected at the light-microscopic level using
the triple-stain technique. Addition of human ANP-(5
28) for 60 min
caused significant enhancements of acrosome reaction of capacitated
human spermatozoa compared with untreated cells (Fig.
1). Maximal response of acrosome reaction
(~2.5-fold) was detected at 1 nM ANP. Human ANP-(1
28), human
ANP-(5
28), and rat ANP-(5
28) showed similar activities. In further
experiments, we utilized human ANP-(5
28).

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Fig. 1.
Dose response for effect of atrial natriuretic peptide (ANP) on human
sperm acrosome reaction. Human semen was washed twice with Ham's F-10
medium containing human serum albumin (HSA, 0.5%) and further
incubated in above medium for capacitation for 2.5 h. Sperm (5 × 108/tube) were then washed again
and incubated for 60 min with increasing concentrations of human
ANP-(5 28) in above medium at 35°C. Acrosome reaction was
determined by triple-stain method. In this and subsequent figures,
results are means ± SE of 3 experiments, each done in triplicate,
and statistical symbols are as follows:
* P < 0.05;
** P < 0.01;
*** P < 0.001 vs. control in
paired Student's t-test.
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The percentage of the acrosome-reacted cells induced by 1 nM ANP was
60-70% of that induced by 2 µM of the
Ca2+ ionophore A-23187 (data not
shown).
A linear fragment of ANP, rat ANP-(13
28), had no effect on acrosome
reaction, indicating that the 17-member disulfide ring is essential for
its biological activity (Fig. 2). Porcine
BNP, another ANP-like compound derived from a different gene (7), had
no effect on the human acrosome reaction at concentrations up to 100 nM
(Fig. 2 and data not shown).

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Fig. 2.
Effect of ANP-like compounds on acrosome reaction in human spermatozoa.
Capacitated human sperm as above were incubated with ANP [human
ANP-(5 28)], porcine brain natriuretic peptide (BNP), or rat
ANP-(13 28), each at 1 nM for 60 min. C, control. Other details as
above (see Fig. 1).
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Role of ANP receptor activation in ANP-induced acrosome reaction.
ANP exerts many of its actions through interaction with the ANP
receptor (ANPR-A), a particulate guanylyl cyclase-linked receptor, and
subsequent generation of cGMP in target cells. The involvement of
ANPR-A in ANP-induced acrosome reaction was tested by a selective ANPR-A antagonist, anantin (17, 29). We found that anantin at 100 nM
completely abolished ANP-induced acrosomal exocytosis of capacitated
human spermatozoa (Fig. 3). The dose of
anantin used here is based on previous observations using bovine sperm cells (31). Anantin by itself had no effect on acrosomal exocytosis or
sperm motility.

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Fig. 3.
Effect of ANP receptor antagonist (ANPR-A) anantin on ANP-induced
acrosome reaction in human spermatozoa. Capacitated human sperm as
above were incubated with ANP (1 nM), anantin (an, 100 nM), or both for
60 min, and acrosome reaction was determined. For other details see
Fig. 1.
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Role of cGMP in ANP-induced acrosome reaction.
Because ANPR-A is a guanylyl cyclase-linked receptor, it was of
interest to analyze the role of cGMP in ANP action. Indeed, Anderson et
al. (2) have recently demonstrated elevation of cGMP by ANP in human
sperm, and we found similar results in bovine sperm (31). Furthermore,
we were able to mimic the stimulatory effect of ANP on the acrosome
reaction in capacitated human spermatozoa by using a membrane-permeable
analog of cGMP, 8-BrcGMP. Indeed, the cGMP analog caused a similar
elevation in acrosome reaction to that obtained with ANP (Fig.
4). These results support the notion that
ANP-induced acrosome reaction is mediated via activation of the
guanylyl cyclase-linked receptor, ANPR-A, and subsequent formation of
cGMP.

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Fig. 4.
Effect of 8-bromo-cGMP (8-BrcGMP) on acrosome reaction in human
spermatozoa. Capacitated human sperm as above were incubated with
increasing doses of permeable cGMP analog 8-BrcGMP for 60 min, and
acrosome reaction was determined. For other details see legend to
Fig.1.
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Role of PKC in ANP stimulation of acrosome reaction.
To evaluate the role of PKC in ANP action, we employed inhibition and
depletion of PKC. Incubation of the cells with increasing concentrations of the PKC inhibitors staurosporine and GF-109203X resulted in a dose-related inhibition of ANP-induced acrosome reaction
(Figs. 5 and
6). In another approach, we employed
PKC-downregulated cells that were obtained by preincubation with TPA
(22). Indeed, incubation of noncapacitated human sperm with TPA (500 ng/ml for 3 h) reduced endogenous PKC enzymatic activity from 0.25 pmol 32P · min
1 · µg
protein
1 to undetectable
levels. ANP-induced acrosome reaction was abolished in the
downregulated cells, supporting the inhibition experiments (Fig.
7). On the other hand, stimulation of
acrosome reaction by 8-BrcGMP was only slightly affected by
downregulation of endogenous PKC (Fig. 8).
The results suggest that cGMP acts downstream, or in parallel to PKC
during ANP action.

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Fig. 5.
Effect of protein kinase C (PKC) inhibitor staurosporine on ANP-induced
acrosome reaction of human spermatozoa. Capacitated human sperm as
above were incubated with increasing doses of staurosporine for 5 min
followed by ANP (1 nM) for 60 min, and acrosome reaction was
determined. For other details see Fig. 1.
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Fig. 6.
Effect of PKC inhibitor GF-109203X on ANP-induced acrosome reaction of
human spermatozoa. Capacitated human sperm as above were incubated with
increasing doses of GF-109203X (GF) for 5 min followed by ANP (1 nM)
for 60 min, and acrosome reaction was determined. For other details see
Fig. 1.
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Fig. 7.
Effect of downregulation of PKC on ANP-induced acrosome reaction.
Noncapacitated human sperm were preincubated with or without
12-O-tetradecanoylphorbol 13-acetate
(TPA, 500 ng/ml for 3 h) to achieve downregulation of endogenous PKC
(DR). Sperm were then washed and further incubated with or without ANP
(1 nM) for 60 min, and acrosome reaction was determined. For other
details see legend to Fig. 1.
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Fig. 8.
Effect of downregulation of PKC on 8-BrcGMP-induced acrosome reaction.
Noncapacitated human sperm were preincubated with or without TPA (500 ng/ml for 3 h) to achieve downregulation of endogenous PKC. Sperm was
then washed and further incubated with or without 8-BrcGMP (1 mM) for
60 min, and acrosome reaction was determined. For other details see
legend to Fig. 1.
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Role of Ca2+
in ANP-induced acrosome reaction.
Acrosomal exocytosis in capacitated human spermatozoa is believed to be
a Ca2+-dependent process (30). We
therefore tested the ANP-induced acrosome reaction to see whether it is
Ca2+ dependent. We incubated
capacitated human spermatozoa in
Ca2+-free medium with or without
ethylene glycol-bis(
-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA). Removal of Ca2+ alone
was not sufficient to block ANP action. On the other hand, removal of
Ca2+ and addition of EGTA
abolished ANP-induced acrosome reaction (Fig
9). The results indicate that ANP-induced
acrosomal exocytosis requires extracellular
Ca2+ influx.

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Fig. 9.
Effect of Ca2+ removal on
ANP-induced acrosome reaction. Capacitated human sperm were incubated
with ANP (1 nM) for 60 min in Ham's F-10 medium + HSA (0.5%), Ham's
F-10 + HSA (0.5%) without Ca2+,
or Ham's F-10 +HSA (0.5%) without
Ca2+ and with EGTA (0.03 mM), and
acrosome reaction was determined. For other details see legend to Fig.
1.
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 |
DISCUSSION |
Multiple physiological agonists probably participate in the regulation
of acrosomal exocytosis (2, 3, 10, 19, 28, 30, 31). Such agonists may
originate from the egg or its associated cellular and acellular
structures or from the female reproductive tract.
ANP could serve as such a physiological agonist. It is synthesized in
granulosa cells (14) and oocytes (15) of mammalian ovaries (8, 13, 14)
and secreted into the follicular fluids. Indeed, ANP is found in the
follicular fluids of human and other species (2, 24, 25). ANP was
reported to play a role in the development of ovarian follicles (27),
in steroidogenesis in ovarian granulosa cells (14), and in the process
of oocyte maturation (27). Cyclic changes in ovarian ANP levels were
observed during the estrous cycle of the rat (13). ANP derived from the ovary may therefore act on spermatozoa and affect their function. Indeed, high-affinity ANPR-A (particulate guanylyl cyclase) has been
localized in human spermatozoa (23). Thus ANP derived from granulosa
cells and/or oocytes may act on spermatozoa before and during
fertilization. ANP-stimulatory actions on mammalian spermatozoa have
been reported in recent studies (2, 5, 31, 32). We have demonstrated
that ANP-induced attraction (chemotaxis) and enhanced swimming speed
(chemokinesis) in human spermatozoa in vitro (32). ANP was also
reported to induce acrosome reaction both in human (2, 5) and bull
spermatozoa (31), apparently by cGMP elevation (2, 31). The results of
the present study further elucidate the mechanisms underlying
ANP-induced acrosome reaction of capacitated human spermatozoa.
Induction of acrosomal exocytosis of capacitated human spermatozoa by
ANP is in good agreement with dissociation constants obtained for ANP
binding to different somatic cell types (7) and human spermatozoa (23) and also with the levels observed in follicular fluids (2). These
observations support a putative physiological role for the hormone in
the regulation of acrosomal exocytosis. ANP may also contribute to
successful fertilization, since Anderson et al. (2) have found that ANP
levels in human follicular fluids are corrrelated with successful in
vitro fertilization. The effect of ANP on human spermatozoa is specific
and mediated by plasma membrane ANPR-A as evident by inhibition of ANP
action by the selective ANPR-A antagonist, anantin.
ANP induces acrosome reaction of human sperm, apparently by
Ca2+ influx, PKC activation, and
cGMP formation. Removal of extracellular Ca2+ had no inhibitory effect on
ANP action (2). This has led Anderson et al. (2) to suggest that ANP
does not require extracellular Ca2+ to exert its stimulatory
response on acrosome reaction. We investigated the role of
Ca2+ further and demonstrate here
inhibition of ANP action by removal of extracellular
Ca2+ and addition of EGTA to
chelate residual Ca2+. We
therefore suggest that ANP-induced acrosome reaction is dependent on
extracellular Ca2+ influx by ANP.
We also found that PKC is involved in ANP-induced acrosome reaction.
The PKC inhibitors, staurosporine and the more selective drug,
GF-109203X, produced a dose-related inhibition of ANP action. Furthermore, downregulation of endogenous PKC by preincubation with TPA
also resulted in inhibition of ANP action. Because previous studies
have shown that PKC-induced acrosome reaction does not depend on extra-
or intracellular Ca2+ levels (22),
we propose that PKC acts downstream to
Ca2+ during ANP action.
The interaction of ANP with the ANPR-A results in cGMP elevation in
bovine and human sperm (2, 31). Indeed, cGMP seems to be involved in
ANP action, since the addition of the permeable analog, 8-BrcGMP,
stimulated acrosome reaction (present results). Furthermore, a
particulate guanylyl cyclase inhibitor, LY-83583, was found capable of
inhibition of ANP action on human sperm acrosome reaction (2). The
results are in agreement with our findings that anantin blocked the
effect of ANP. To analyze the site of cGMP action in relation to PKC,
we used downregulated cells. As mentioned above, downregulation of
endogenous PKC abolished the effect of ANP but had no significant
effect on 8-BrcGMP. The results suggest that cGMP acts independent to
or downstream to PKC during ANP action on human sperm acrosome
reaction.
Our proposed signaling for ANP action seems to differ from that
observed in renal glomerular cells, in which ANP was reported to
antagonize the PKC signal (4). Nevertheless, ANP was reported to
stimulate phosphoinositide turnover in bovine aortic smooth muscle
cells (9), and PKC was shown to mediate
Ca2+-induced activation of rat
colonic particulate guanylyl cyclase (12). We cannot rule out the
possibility that ANP also binds to a second receptor subtype such as
ANP clearance receptor (ANPR-C) (1), which is coupled to
phosphoinositide turnover (1, 9). In this case,
Ca2+ and PKC might be activated
via ANPR-C, whereas cGMP is formed via ANPR-A. We therefore have to
assume that ANP-induced acrosome reaction is dependent on all three
messenger molecules, which act in parallel. The possibility that cGMP
is responsible for Ca2+ elevation
and PKC activation via enhanced phosphoinositide turnover (18) is less
likely. In sperm cells, cGMP is thought to inhibit phosphoinositide
turnover (1). In addition, downregulation of PKC did not block
8-BrcGMP-induced acrosome reaction, indicating that cGMP acts
downstream or in parallel to PKC.
Our results suggest that ANP may join other sperm ligands, such as zona
pellucida glycoproteins (e.g., ZP-3) and progesterone, in eliciting
acrosome reaction of human sperm.
 |
ACKNOWLEDGEMENTS |
Ronit Rotem and Nadav Zamir contributed equally to this work.
 |
FOOTNOTES |
The study was supported by a grant from the Ministry of Health.
Address for reprint requests: Z. Naor, Dept. of Biochemistry, Tel Aviv
University, Ramat Aviv 69978, Israel.
Received 9 June 1997; accepted in final form 25 August 1997.
 |
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