Departments of Obstetrics and Gynecology and Cell Biology, Vanderbilt
University School of Medicine, Room D-3243 MCN, Nashville, TN 37232-2633,
USA
* Present address: Research Center of Molecular Biology, Central South
University, Xiang Ya School of Medicine, Changsha, Hunan 410078, Peoples
Republic of China
Author for correspondence (e-mail:
daulat.tulsiani{at}vanderbilt.edu)
Accepted 28 January 2003
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Summary |
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Key words: Sperm capacitation, Protein tyrosine phosphorylation, Mammalian spermatozoa, Calmodulin, Calmodulin antagonists, Sperm mobility
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Introduction |
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All mammalian spermatozoa studied thus far undergo capacitation after
residing in the female genital tract for a certain period of time
(Yanagimachi and Chang, 1963;
Yanagimachi, 1994
). Sperm
cells can also be capacitated in vitro by incubating in a chemically defined
medium supplemented with serum albumin, usually BSA
(Dow and Bavister, 1989
), or
methyl-ß-cyclodextrin (Visconti et
al., 1999
) and energy substrates, such as glucose and pyruvate as
well as reagents used in the Krebs-Ringer bicarbonate medium. It should be
noted that albumin is a major protein both in the female genital tract and in
the in vitro capacitation medium. The protein is believed to facilitate
capacitation by efflux of sterols (mainly cholesterol) from the sperm PM
(Cross, 1998
;
Visconti et al., 1999
). The in
vivo/in vitro removal of sterols is believed to increase fluidity and
permeability of the sperm PM, initiating capacitation.
It is important to emphasize that capacitation is correlated with protein
tyrosine phosphorylation of a subset of sperm molecules
(Visconti and Kopf, 1998;
Visconti et al., 1999
) as well
as with hyperactivated motility (Fraser,
1995
). Although, in some instances, changes in sperm motility
pattern (hyperactivity) and capacitation can be separated, the two events are
generally considered dependent since one of the features of sperm capacitation
is hyperactivated mobility (Yanagimachi,
1994
). Capacitation, therefore, is the net result of changes that
occur (1) on the sperm head, which enables it to bind to the extracellular
coat of the egg, the zona pellucida, and undergo the acrosome reaction (AR)
and (2) in the flagellum, which facilitates hyperactivated sperm motility.
In a previous report (Bendahmane et al.,
2001), we demonstrated the functional significance of calmodulin
(CaM), a 17 kDa Ca2+-binding protein, in capacitation and in the
neoglycoprotein-/zona pellucida-induced AR. To characterize further the
involvement of CaM in capacitation-associated protein tyrosine
phosphorylation, we have examined the effect of CaM antagonists on the
biochemical changes in capacitating spermatozoa. Data included in this report
demonstrate that CaM inhibitors differentially affect capacitation-associated
protein tyrosine phosphorylation of a subset of sperm components, and
hyperactivated motility.
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Materials and Methods |
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Preparation and use of CaM antagonists
A 100-fold concentrated stock solution was prepared either in distilled
water (compound 48/80, CBD, W7, and W13), methanol (OA), or 10% DMSO (CZ).
Aliquots of the stock solution were mixed with the sperm suspension to achieve
the desired concentration. In experiments where antagonists were solubilized
in DMSO or methanol, the sperm cells were incubated concurrently with the
solvent as a control. The final concentration of DMSO and methanol was 0.1%
and 1%, respectively. At these concentrations, the organic solvents had no
effect on capacitation, the AR, sperm motility, cell viability, or protein
tyrosine phosphorylation.
Preparation and capacitation of the cauda spermatozoa
Cauda epididymidis was excised and freed of the fat-pad, blood vessels and
connective tissue under a dissecting microscope. The tissue was transferred to
a 3 ml Petri dish containing 1 ml EKRB medium pre-warmed to room temperature
(24°C), and cut in several places with iridectomy scissors to release the
spermatozoa into the medium. After 5 minutes, the sperm suspension was
transferred to a 15 ml centrifuge tube. The concentration of the spermatozoa
dispersed in the medium was assessed using a Neubauer hemocytometric chamber.
The sperm concentration was adjusted to 1x106/ml by adding
EKRB medium containing 6 mg BSA/ml (the final concentration of BSA was 3
mg/ml) or 2 mM methyl-ß-cyclodextrin (the final concentration of
cyclodextrin was 1 mM) and subjected to capacitation in the absence or
presence of CaM antagonists or purified CaM by incubation at 37°C under 5%
CO2 in air as described
(Bendahmane et al., 2001).
Sperm motility
Spermatozoa were examined by phase-contrast microscopy, and the percentage
of motile spermatozoa (a combined measure of flagellum beat and forward
motility) was determined by scoring 100 spermatozoa.
Sperm viability
Sperm samples (300 µl) incubated in the absence (control) or presence
(experimental) of CaM antagonist were stained at 36°C with 12 µM
propidium iodide for 15 minutes as described
(Garner and Johnson, 1995).
Spermatozoa (200) were observed and counted under
phase-contrast/epifluorescene illumination with a Zeiss Axiophot
photomicroscope, and the percentage of dead (red stain on the nucleus) and
live (nonstained) spermatozoa were calculated.
Assessment of acrosomal status
The status of the sperm acrosome was assessed using the Coomassie brilliant
blue G-250 dye method of Larson and Miller
(Larson and Miller, 1999) as
described (Bendahmane et al.,
2001
). Spermatozoa (200) were scored in duplicate, and the
percentage of spermatozoa that had undergone the AR was calculated.
Extraction of spermatozoa
On the completion of incubation, aliquots were withdrawn for the assessment
of sperm motility and sperm viability. The remaining spermatozoa were pelleted
by centrifugation at 5000 g for 1 minute The supernatant was
discarded and pelleted spermatozoa were re-suspended in 0.5 ml PBS and
centrifuged as above. The sperm pellet was suspended in 50 µl Laemmli's
sample buffer (Laemmli, 1970)
without 2-mercaptoethanol and placed in boiling water for 5 minutes. Following
extraction, the sperm suspension was centrifuged at 5000 g for
3 minutes. The supernatant was removed by aspiration, transferred to a new
tube, and 2-mercaptoethanol was added to a final concentration of 5% as
described (Visconti et al.,
1999
). The sperm extract was either used immediately or stored
frozen at 70°C.
SDS-PAGE and immunoblotting
The extract from 5x105 spermatozoa was resolved on 1.5
mm-thick polyacrylamide gel (10%) along with high-molecular-weight marker
proteins as described (Tulsiani et al.,
1995). The resolved polypeptides were electrophoretically
transferred to a nitrocellulose membrane by the method of Towbin et al.
(Towbin et al., 1979
), as
described (Tulsiani et al.,
1995
). Following the transfer, the membrane was blocked for 1 hour
in Tris-buffered saline (TBS; 20 mM Tris base, 137 mM NaCl, pH 7.6) containing
0.1% Tween 20 and 30 mg BSA/ml at 25°C, and incubated with monoclonal
anti-phosphotyrosine antibody PY20 at 1:5000 dilution for 1 hour at room
temperature. After this incubation, the membrane was washed for 30 minutes in
TBS/0.1% Tween 20 (once for 15 minutes and three times for 5 minutes each),
followed by incubation with anti-mouse IgG-HRP conjugate at 1:3000 dilution
for 1 hour. The membrane was washed for 30 minutes as above and the
protein-tyrosine-phosphorylated components were revealed using the ECL kit
according to the manufacturer's instructions. Tyrosine-phosphorylated bands
were scanned and their intensities quantified using IPLab Gel H Program.
Statistical analysis
The results are presented as mean±s.d.; means of control and
experimental groups were compared using the Newman-Keuls test after one-way
ANOVA to determine statistically significant differences
(P<0.05).
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Results |
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|
|
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Effects of CaM antagonists on capacitation-associated sperm protein
tyrosine phosphorylation
In preliminary studies, we incubated cauda epididymal spermatozoa in EKRB
medium supplemented with 3 mg BSA/ml or 1 mM methyl-ß-cyclodextrin in the
absence or presence of six CaM antagonists for 60 minutes and 90 minutes at
37°C under 5% CO2 in air. Interestingly, three of the six
antagonists used (W7, OA and CZ) showed a significant inhibitory effect on the
tyrosine phosphorylation of all sperm components
(Fig. 2A). By contrast, the
other three CaM antagonists (compound 48/80, W13 and CBD) had no effect on
tyrosine phosphorylation of sperm (Fig.
3A). These incubations were carried out for 90 minutes, the time
needed for optimal levels of protein tyrosine phosphorylation. Data presented
in Figs 2A and
3A demonstrate differential
affects of the two sets of CaM antagonists on protein tyrosine phosphorylation
of the mouse sperm molecules.
Since the presence of BSA or methyl-ß-cyclodextrin in the capacitation medium had similar effects on sperm protein tyrosine phosphorylation, it was of interest to determine the effect of CaM antagonists on tyrosine phosphorylation of the sperm molecules. Spermatozoa were capacitated in EKRB medium supplemented with methyl-ß-cyclodextrin (instead of BSA) in the absence or presence of CaM antagonist. After 90 minutes of incubation, sperm cells were extracted, electrophoresed, transferred to a nitrocellulose membrane and the phosphorylated bands were revealed as above. Data demonstrate that tyrosine phosphorylation of sperm proteins, capacitated either in medium supplemented with BSA (Fig. 2A; Fig. 3A) or methyl-ß-cyclodextrin (Fig. 2B; Fig. 3B) responded to the CaM antagonists in a similar manner.
Do CaM antagonists have an adverse effect on sperm motility and sperm
viability?
Our next approach was to examine whether CaM inhibitors have any effect on
sperm viability and sperm motility. Spermatozoa were capacitated in EKRB
medium supplemented with 3 mg BSA/ml with or without the known concentration
of CaM antagonists. After 90 minutes incubation at 37°C in 5%
CO2 in air, aliquots were checked for sperm motility and sperm
viability. The remaining cells were pelleted and used to examine protein
tyrosine phosphorylation by the procedures described above. Data presented in
Table 2 demonstrate that,
whereas OA, W7 and CZ inhibited/prevented sperm motility and protein tyrosine
phosphorylation, the other three antagonists (compound 48/80, W13 and CBD) had
no effect on these parameters (Table
2). Furthermore, OA, W7 and CZ inhibited/prevented motility
without affecting sperm viability, as monitored by propidium iodine staining;
approximately 50% of the sperm cells remained viable after 90 minutes of
incubation in the presence or absence of these antagonists.
|
Purified CaM increases tyrosine phosphorylation of two sperm
proteins
In a previous report (Bendahmane, 2001), we demonstrated that the inclusion
of purified CaM in the incubation medium largely reversed the AR-blocking
effects of antagonist during in vitro sperm capacitation. Furthermore, our
group has recently reported that the inclusion of purified CaM in in vitro
incubation medium did not alter the overall rate of capacitation; however, its
presence accelerated initial stages of capacitation-associated membrane
primings (Abou-Haila and Tulsiani,
2002), a result consistent with the suggestion that CaM has a role
in sperm capacitation. In this study, we have attempted to assess the role of
CaM in protein tyrosine phosphorylation of sperm molecules. Mouse spermatozoa
were incubated in the capacitation medium in the absence or presence of 10
µM purified CaM. Following this incubation, the sperm cells were pelleted,
extracted, electrophoresed and the tyrosine-phosphorylated bands were
revealed. The bands present in the control and experimental groups were
scanned and quantitated by densitometric analyses. Each band in the control
group was considered 100% and the relative intensity of the same molecular
weight band in the experimental group was calculated. Data presented in
Fig. 4 demonstrate that CaM
increased capacitation-associated tyrosine phosphorylation of all molecules;
however, only two (95 kDa and 82 kDa) showed statistically significant
increases.
|
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Discussion |
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Several recent studies have demonstrated that in vitro capacitation of
spermatozoa from several species is also associated with increased protein
tyrosine phosphorylation of a subset of macromolecules
(Visconti et al., 1995a;
Visconti et al., 1999
). In
this study, we have used a pharmacological approach in an attempt to
characterize further the inter-relationship between capacitation and protein
tyrosine phosphorylation. First, we confirmed published reports by
demonstrating that there was indeed a time-dependent increase in tyrosine
phosphorylation of a subset of molecules when the mouse spermatozoa were
incubated in a medium that favors capacitation
(Fig. 1).
The capacitation-associated tyrosine phosphorylation of the sperm
components has been reported to be dependent on Ca2+ and
NaHCO3. The concentration of these components needed in the
incubation medium for the protein tyrosine phosphorylation to occur was
reported to be correlated with that needed for sperm capacitation, a result
consistent with the suggestion that tyrosine phosphorylation of sperm proteins
and sperm capacitation are highly correlated
(Visconti et al., 1995a;
Visconti et al., 1999
). Thus,
our next goal was to determine the inter-relationship between capacitation and
protein tyrosine phosphorylation. This was accomplished by incubating the
cauda epididymal spermatozoa in EKRB, supplemented with BSA or
methyl-ß-cyclodextrin, in the absence and presence of several CaM
antagonists, and examining sperm capacitation and protein tyrosine
phosphorylation. The six CaM inhibitors used in two previous reports
(Bendahmane et al., 2001
;
Bendahmane et al., 2002
) and in
the present study (Table 1)
were effective in inhibiting/preventing sperm capacitation as evident by their
poor response to the agonist-induced AR. Three of the six antagonists used
(W7, OA and CZ) inhibited/prevented capacitation
(Table 1), sperm motility
(Table 2), and tyrosine
phosphorylation of all sperm components
(Fig. 2). However, the effect
of these inhibitors varies, with OA and W7 having a greater effect on motility
and tyrosine phosphorylation than CZ (Table
2). The adverse effect of these antagonists on sperm motility was
not due to altered cell viability, as assessed by the propidium iodine
protocol, since the sperm cells in the absence or presence of CaM antagonists
displayed similar viability (Table
2). By contrast, the other three CaM antagonists (compound 48/80,
W13 and CBD) inhibited capacitation (Table
1) without adversely affecting sperm motility or cell viability
(Table 2) or protein tyrosine
phosphorylation (Fig. 3). Taken
together, data from the two sets of CaM antagonists suggest that they
inhibit/prevent capacitation by at least two regulatory mechanisms
(Fig. 5). The first event might
adversely affect sperm motility before inhibiting protein tyrosine
phosphorylation (Fig. 5A) or
vice versa (Fig. 5B). The
second event could be inhibition/prevention of capacitation without affecting
sperm motility or protein tyrosine phosphorylation
(Fig. 5). These data imply that
all CaM antagonists used in this study act on the sperm head
(inhibiting/preventing capacitation as evident by the failure of the sperm to
respond to agonists), but only W7, OA and CZ have an adverse effect on the
sperm tail (Table 2). These
results suggest a close inter-relationship between sperm protein tyrosine
phosphorylation and motility. The proposed relationship is consistent with the
experimental evidence from many investigators suggesting that protein tyrosine
phosphorylation plays an important role in the control of the hyperactivated
motility (Vijayaraghavan et al.,
1997
; Si and Okuno,
1999
; Holt and Harrison,
2002
). Our data, however, do not allow us to conclude whether
protein tyrosine phosphorylation precedes motility or vice versa.
|
The fact that tyrosine phosphorylation and sperm capacitation are
stimulated by cAMP analogues, but inhibited by cAMP antagonists or protein
kinase A (PKA) inhibitors, strongly suggests that the cAMP/PKA signaling
pathways are involved in both processes
(Galantino-Homer et al., 1997;
Visconti et al., 1999
;
Leclerc et al., 1996
). Further
support for the role of cAMP-dependent protein tyrosine phosphorylation during
sperm capacitation comes from the observation that calyculin A, an inhibitor
of protein phosphatases 1 and 2A (Ishihara
et al., 1989
), enhances protein phosphorylation and sperm
capacitation (Leclerc et al.,
1996
; Furuya, 1993). The present pharmacological approach
demonstrates that a set of CaM antagonists (compound 48/80, W13 and CBD) can
inhibit/prevent capacitation without adversely affecting sperm motility or
protein tyrosine phosphorylation. These data raise questions about the
proposed tight correlation between the tyrosine phosphorylation and
capacitation of mouse spermatozoa. Future studies should be directed to
determine whether the antagonists that affected both processes have additional
molecular targets beyond CaM. New data will provide insights into the
mechanisms underlying protein tyrosine phosphorylation and sperm
capacitation.
Mammalian spermatozoa contain two main parts: (1) the head with the
acrosomal (anterior head) and post-acrosomal (posterior head) regions, and (2)
the flagellum (Yanagimachi,
1994; Tulsiani and Abou-Haila,
2001
). Whereas the receptor(s) responsible for binding to the ZP
and initiating a signal transduction cascade prior to induction of the AR are
present on the anterior head region of the capacitated spermatozoa
(Tulsiani and Abou-Haila,
2001
), the hyperactivated motility is a net result of molecular
changes on the flagellum (Yanagimachi,
1970
; Yanagimachi,
1994
; Suarez,
1996
). Since all antagonists used in this study
inhibited/prevented capacitation, it is reasonable to suggest that their
blocking effects are due to inhibition of CaM in the sperm head. However, why
only W7, OA and CZ adversely affected sperm motility and protein tyrosine
phosphorylation or why OA and W7 (but not CZ) nearly blocked motility is not
known at the present time. One possibility is that these three reagents might
not elicit their effects through CaM alone, but have other molecular targets
such as FSP95 (Mandal et al.,
1999
), a testis-specific fibrous-sheath protein. Alternatively,
perhaps only some of the reagents are able to enter the tail and block CaM.
Additional studies will be needed to resolve these issues.
It should be noted that in vitro capacitation requires BSA
(Dow and Bavister, 1989) or
methyl-ß-cyclodextrin (Visconti et
al., 1999
) in the incubation medium. The two reagents are thought
to promote capacitation through their ability to efflux cholesterol from the
sperm PM (Visconti et al.,
1999
). The loss of cholesterol increases fluidity and permeability
of the sperm PM, initiating the signaling events leading to capacitation.
Since the two sets of CaM antagonists have similar effects on BSA- or
methyl-ß-cyclodextrin-supplemented medium during in vitro capacitation,
it is reasonable to conclude that these antagonists inhibit/prevent
capacitation in these media by blocking the same signaling pathway. It would
be interesting to determine if CaM antagonists inhibit/prevent capacitation by
blocking the loss of cholesterol from the sperm membrane. Data from this
approach will provide new insights into the inter-relationship between the
loss of cholesterol, protein tyrosine phosphorylation and sperm
capacitation.
How does CaM influence protein tyrosine phosphorylation? The acidic protein
has been reported to stimulate adenylyl cyclase
(Gross et al., 1987;
Kopf and Vacquier, 1984
), a PM
enzyme responsible for the synthesis of cAMP. Thus, it seems likely that the
three antagonists that inhibit/prevent the protein tyrosine phosphorylation do
so by their inhibitory effect on CaM-dependent adenylyl cyclase activity. The
inhibition of this enzyme activity is expected to start a chain reaction by
reducing levels of cAMP and inhibiting the proposed cross-talk between cAMP
and tyrosine kinase second messenger systems
(Visconti and Kopf, 1998
),
thereby inhibiting protein phosphorylation and sperm capacitation. As a
corollary, it seems likely that the inclusion of purified CaM in the
capacitation medium will stimulate adenylyl cyclase, which in turn will
increase the levels of cAMP. The increased cAMP levels, suggested to act
upstream on the protein tyrosine phosphorylation
(Visconti et al., 1995b
;
Visconti and Kopf, 1998
),
might be important for the observed increases in protein tyrosine
phosphorylation of the 95 kDa and 82 kDa sperm molecules. Whether the 95 kDa
component is a unique hexokinase (Kalab et
al., 1994
), FSP95 (Mandal et
al., 1999
) or has both ZP3-binding and a tyrosine kinase activity
(Leyton and Saling, 1989
) is
not yet known.
Does CaM enter the capacitating spermatozoa and trigger intracellular
signaling that promotes the AR? A published report provided evidence
suggesting that inclusion of pure CaM in the culture medium promotes DNA
synthesis and cell proliferation in human leukemia lymphocytes
(Crocker et al., 1988). These
data strongly suggest that CaM can influence mitosis through an extracellular
mechanism. Furthermore, extracellular CaM was reported to inhibit monocyte
tumor necrosis factor release and augment neutrophil elastase release
(Houston et al., 1997
). These
data, in conjunction with the reported occurrence of receptor(s) on monocytic
cell lines, strongly suggest that CaM possesses an extracellular signaling
role in addition to its intracellular regulatory functions
(Houston et al., 1997
).
Whether CaM functions via sperm surface receptors is not yet known.
The differential effects of the two sets of CaM antagonists on sperm
capacitation and protein tyrosine phosphorylation raise many interesting
questions on the inter-relationship between sperm cholesterol, protein
tyrosine phosphorylation and capacitation. Cross has reported that, when human
spermatozoa are incubated in capacitating medium in vitro, there is a gradual
loss of cholesterol; however, the acrosomal responsiveness does not develop
for some time (Cross, 1998).
This result is consistent with the author's suggestion that cholesterol loss
precedes capacitation. It would be interesting to see if CaM acts upstream or
downstream to the cholesterol efflux or if there are regional modifications of
the sperm membranes.
In summary, this work uses a pharmacological approach to examine the inter-relationship between motility, tyrosine phosphorylation of sperm components and sperm capacitation. Our data provide evidence suggesting the occurrence of multiple pathways that regulate sperm motility, protein tyrosine phosphorylation and sperm capacitation. It would be interesting to use CaM inhibitors to determine whether the loss of cholesterol and sperm capacitation is tightly correlated. Data from these studies will allow new insights for blocking capacitation and altering sperm function.
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Acknowledgments |
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